1. Jensen, M., Ahlberg Gagner, V., Cabello Sanchez, J., Bengtsson, A. U. J., Ekstrom, J. C., Bjorg Ulfarsdottir, T., Garcia-Bonete, M.-J., Jurgilaitis, A., Kroon, D., Pham, V. T., Checcia, S., Coudert-Alteirac, H., Schewa, S., Rossle, M., Rodilla, H., Stake, J., Zhaunerchyk, V., Larsson, J. & Katona, G. (2021) High-resolution macromolecular crystallography at the FemtoMAX beamline with time-over-threshold photon detection. J. Synchrotron Radiat., 28(1), 64-70. [FULLTEXT
    Protein dynamics contribute to protein function on different time scales. Ultrafast X-ray diffraction snapshots can visualize the location and amplitude of atom displacements after perturbation. Since amplitudes of ultrafast motions are small, high-quality X-ray diffraction data is necessary for detection. Diffraction from bovine trypsin crystals using single femtosecond X-ray pulses was recorded at FemtoMAX, which is a versatile beamline of the MAX IV synchrotron. The time-over-threshold detection made it possible that single photons are distinguishable even under short-pulse low-repetition-rate conditions. The diffraction data quality from FemtoMAX beamline enables atomic resolution investigation of protein structures. This evaluation is based on the shape of the Wilson plot, cumulative intensity distribution compared with theoretical distribution, I/σ, Rmerge/Rmeas and CC1/2 statistics versus resolution. The FemtoMAX beamline provides an interesting alternative to X-ray free-electron lasers when studying reversible processes in protein crystals.


  2. Dods, R., Bath, P., Morozov, D., Gagner, V. A., Arnlund, D., Luk, H. L., Kubel, J., Maj, M., Vallejos, A., Wickstrand, C., Bosman, R., Beyerlein, K. R., Nelson, G., Liang, M., Milathianaki, D., Robinson, J., Harimoorthy, R., Berntsen, P., Malmerberg, E., Johansson, L., Andersson, R., Carbajo, S., Claesson, E., Conrad, C. E., Dahl, P., Hammarin, G., Hunter, M. S., Li, C., Lisova, S., Royant, A., Safari, C., Sharma, A., Williams, G. J., Yefanov, O., Westenhoff, S., Davidsson, J., DePonte, D. P., Boutet, S., Barty, A., Katona, G., Groenhof, G., Branden, G. & Neutze, R. (2020) Ultrafast structural changes within a photosynthetic reaction centre. Nature, 589(7841), 310-14. [FULLTEXT
    Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography1 using an X-ray free-electron laser2 to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions.

  3. Schewa, S., Schroer, M. A., Zickmantel, T., Song, Y. H., Blanchet, C. E., Gruzinov, A. Y., Katona, G., Svergun, D. I. & Roessle, M. (2020) A tool for visualizing protein motions in time-resolved crystallography. Rev. Sci. Instrum., 91(8), 084101. [FULLTEXT
    Excitation frequencies in the terahertz (THz) range are expected to lead to functionally relevant domain movements within the biological macromolecules such as proteins. The possibility of examining such movements in an aqueous environment is particularly valuable since here proteins are not deprived of any motional degrees of freedom. Small angle x-ray scattering (SAXS) is a powerful method to study the structure and domain movements of proteins in solution. Here, we present a microfluidic cell for SAXS experiments, which is also transparent for THz radiation. Specifically, cell dimensions and material were optimized for both radiation sources. In addition, the polystyrene cell can be 3D printed and easily assembled. We demonstrate the practicality of our design for SAXS measurements on several proteins in solution.

  4. Wickstrand, C., Katona, G., Nakane, T., Nogly, P., Standfuss, J., Nango, E. & Neutze, R. (2020) A tool for visualizing protein motions in time-resolved crystallography. Structural Dynamics, 7(2), 024701. [FULLTEXT
    Time-resolved serial femtosecond crystallography (TR-SFX) at an x-ray free electron laser enables protein structural changes to be imaged on time-scales from femtoseconds to seconds. It can, however, be difficult to grasp the nature and timescale of global protein motions when structural changes are not isolated near a single active site. New tools are, therefore, needed to represent the global nature of electron density changes and their correlation with modeled protein structural changes. Here, we use TR-SFX data from bacteriorhodopsin to develop and validate a method for quantifying time-dependent electron density changes and correlating them throughout the protein. We define a spherical volume of difference electron density about selected atoms, average separately the positive and negative electron difference densities within each volume, and walk this spherical volume through all atoms within the protein. By correlating the resulting difference electron density amplitudes with time, our approach facilitates an initial assessment of the number and timescale of structural intermediates and highlights quake-like motions on the sub-picosecond timescale. This tool also allows structural models to be compared with experimental data using theoretical difference electron density changes calculated from refined resting and photo-activated structures.

  5. Nass, K., Redecke, L., Perbandt, M., Yefanov, O., Klinge, M., Koopmann, R., Stellato, F., Gabdulkhakov, A., Schonherr, R., Rehders, D., Lahey-Rudolph, J. M., Aquila, A., Barty, A., Basu, S., Doak, R. B., Duden, R., Frank, M., Fromme, R., Kassemeyer, S., Katona, G., Kirian, R., Liu, H., Majoul, I., Martin-Garcia, J. M., Messerschmidt, M., Shoeman, R. L., Weierstall, U., Westenhoff, S., White, T. A., Williams, G. J., Yoon, C. H., Zatsepin, N., Fromme, P., Duszenko, M., Chapman, H. N. & Betzel, C. (2020) In cellulo crystallization of Trypanosoma brucei IMP dehydrogenase enables the identification of genuine co-factors. Nature communications, 11, 620. [FULLTEXT
    Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5'-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 angstrom resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds. Trypanosoma brucei inosine-5 '-monophosphate dehydrogenase (IMPDH) is an enzyme in the guanine nucleotide biosynthesis pathway and of interest as a drug target. Here the authors present the 2.8 angstrom room temperature structure of TbIMPDH determined by utilizing X-ray free-electron laser radiation and crystals that were grown in insect cells and find that ATP and GMP are bound at the canonical sites of the Bateman domains.

  6. Gagnér, V. A., Lundholm, I., Garcia-Bonete, M.-J., Rodilla, H., Friedman, R., Zhaunerchyk, V., Bourenkov, G., Schneider, T., Stake, J. & Katona, G. (2019) Clustering of atomic displacement parameters in bovine trypsin reveals a distributed lattice of atoms with shared chemical properties. Scientific reports, 9, 19281. [FULLTEXT
    Low-frequency vibrations are crucial for protein structure and function, but only a few experimental techniques can shine light on them. The main challenge when addressing protein dynamics in the terahertz domain is the ubiquitous water that exhibit strong absorption. In this paper, we observe the protein atoms directly using X-ray crystallography in bovine trypsin at 100 K while irradiating the crystals with 0.5 THz radiation alternating on and off states. We observed that the anisotropy of atomic displacements increased upon terahertz irradiation. Atomic displacement similarities developed between chemically related atoms and between atoms of the catalytic machinery. This pattern likely arises from delocalized polar vibrational modes rather than delocalized elastic deformations or rigid-body displacements. The displacement correlation between these atoms were detected by a hierarchical clustering method, which can assist the analysis of other ultra-high resolution crystal structures. These experimental and analytical tools provide a detailed description of protein dynamics to complement the structural information from static diffraction experiments.

  7. Garcia-Bonete, M.-J. & Katona, G. (2019) Bayesian machine learning improves single-wavelength anomalous diffraction phasing. Acta Cryst. A., 75, 851-60. [FULLTEXT
    Single-wavelength X-ray anomalous diffraction (SAD) is a frequently employed technique to solve the phase problem in X-ray crystallography. The precision and accuracy of recovered anomalous differences are crucial for determining the correct phases. Continuous rotation (CR) and inverse-beam geometry (IBG) anomalous data collection methods have been performed on tetragonal lysozyme and monoclinic survivin crystals and analysis carried out of how correlated the pairs of Friedel's reflections are after scaling. A multivariate Bayesian model for estimating anomalous differences was tested, which takes into account the correlation between pairs of intensity observations and incorporates the a priori knowledge about the positivity of intensity. The CR and IBG data collection methods resulted in positive correlation between I(+) and I(−) observations, indicating that the anomalous difference dominates between these observations, rather than different levels of radiation damage. An alternative pairing method based on near simultaneously observed Bijvoet's pairs displayed lower correlation and it was unsuccessful for recovering useful anomalous differences when using the multivariate Bayesian model. In contrast, multivariate Bayesian treatment of Friedel's pairs improved the initial phasing of the two tested crystal systems and the two data collection methods.

  8. Garcia-Bonete, M.-J., Jensen, M., & Katona, G. (2019) A practical guide to developing virtual and augmented reality exercises for teaching structural biology. Biochem. Mol. Biol. Edu., 47, 16-24. [FULLTEXT
    Although virtual and augmented reality (VR and AR) techniques have been used extensively in specialized laboratories, only recently did they become affordable, reaching wider consumer markets. With increased availability, it is timely to examine the roles that VR and AR may play in teaching structural biology and in experiencing complex data sets such as macromolecular structures. This guide is suitable for those teachers of structural biology who do not have a deep knowledge of information technologies. This study focuses on three questions: 1) How can teachers of structural biology produce and disseminate VR/AR‐ready educational material with established and user‐friendly software tools?; 2) What are the positive and negative experiences reported by test participants when performing identical learning tasks in the VR and AR environments?; and 3) How do the test participants perceive prerecorded narration during VR/AR exploration?

  9. Garcia-Bonete, M.-J., Jensen, M., Recktenwald, C. V., Rocha, S., Stadler, V., Bokarewa, M. & Katona, G. (2017) Bayesian Analysis of MicroScale Thermophoresis Data to Quantify Affinity of Protein: Protein Interactions with Human Survivin. Scientific reports, 7(1), 16816. [FULLTEXT
    A biomolecular ensemble exhibits different responses to a temperature gradient depending on its diffusion properties. MicroScale Thermophoresis technique exploits this effect and is becoming a popular technique for analyzing interactions of biomolecules in solution. When comparing affinities of related compounds, the reliability of the determined thermodynamic parameters often comes into question. The thermophoresis binding curves can be assessed by Bayesian inference, which provides a probability distribution for the dissociation constant of the interacting partners. By applying Bayesian machine learning principles, binding curves can be autonomously analyzed without manual intervention and without introducing subjective bias by outlier rejection. We demonstrate the Bayesian inference protocol on the known survivin:borealin interaction and on the putative protein-protein interactions between human survivin and two members of the human Shugoshin-like family (hSgol1 and hSgol2). These interactions were identified in a protein microarray binding assay against survivin and confirmed by MicroScale Thermophoresis.

  10. Gravina, G., Wasén, C., Garcia-Bonete, M.-J., Turkkila, M., Erlandsson, M.C., Töyrä Silfverswärd, S., Brisslert, M., Pullerits, R., Andersson, K.M., Katona, G. & Bokarewa, M.I. (2017) Survivin in autoimmune diseases. Autoimmun Rev., 16(8), 845-855. [FULLTEXT
    Survivin is a protein functionally important for cell division, apoptosis, and possibly, for micro-RNA biogenesis. It is an established marker of malignant cell transformation. In non-malignant conditions, the unique properties of survivin make it indispensable for homeostasis of the immune system. Indeed, it is required for the innate and adaptive immune responses, controlling differentiation and maintenance of CD4+ and CD8+ memory T-cells, and in B cell maturation. Recently, survivin has emerged as an important player in the pathogenesis of autoimmune diseases. Under the conditions of unreserved inflammation, survivin enhances antigen presentation, maintains persistence of autoreactive cells, and supports production of autoantibodies. In this context, survivin takes its place as a diagnostic and prognostic marker in rheumatoid arthritis, psoriasis, systemic sclerosis and pulmonary arterial hypertension, neuropathology and multiple sclerosis, inflammatory bowel diseases and oral lichen planus. In this review, we summarise the knowledge about non-malignant properties of survivin and focus on its engagement in cellular and molecular pathology of autoimmune diseases. The review highlights utility of survivin measures for clinical applications. It provides rational for the survivin inhibiting strategies and presents results of recent reports on survivin inhibition in modern therapies of cancers and autoimmune diseases.

  11. Ecsédi, P., Kiss, B., Gógl, G., Radnai, L., Buday, L., Koprivanacz, K., Liliom, K., Leveles, I., Vértessy, B., Jeszenői, N., Hetényi, C., Schlosser, G., Katona, G. & Nyitray, L. (2017) Regulation of the Equilibrium between Closed and Open Conformations of Annexin A2 by N-Terminal Phosphorylation and S100A4-Binding. Structure, 25(8), 16816. [FULLTEXT
    Annexin A2 (ANXA2) has a versatile role in membrane-associated functions including membrane aggregation, endo- and exocytosis, and it is regulated by post-translational modifications and protein-protein interactions through the unstructured N-terminal domain (NTD). Our sequence analysis revealed a short motif responsible for clamping the NTD to the C-terminal core domain (CTD). Structural studies indicated that the flexibility of the NTD and CTD are interrelated and oppositely regulated by Tyr24 phosphorylation and Ser26Glu phosphomimicking mutation. The crystal structure of the ANXA2-S100A4 complex showed that asymmetric binding of S100A4 induces dislocation of the NTD from the CTD and, similar to the Ser26Glu mutation, unmasks the concave side of ANXA2. In contrast, pTyr24 anchors the NTD to the CTD and hampers the membrane-bridging function. This inhibition can be restored by S100A4 and S100A10 binding. Based on our results we provide a structural model for regulation of ANXA2-mediated membrane aggregation by NTD phosphorylation and S100 binding.

  12. Sharma, A., Johansson, L., Dunevall, E., Wahlgren, W.Y., Neutze, R. & Katona, G. (2017) Asymmetry in serial femtosecond crystallography data. Acta Cryst. A, 73(2), 93-101. [FULLTEXT
    Serial crystallography is an increasingly important approach to protein crystallography that exploits both X-ray free-electron laser (XFEL) and synchrotron radiation. Serial crystallography recovers complete X-ray diffraction data by processing and merging diffraction images from thousands of randomly oriented non-uniform microcrystals, of which all observations are partial Bragg reflections. Random fluctuations in the XFEL pulse energy spectrum, variations in the size and shape of microcrystals, integrating over millions of weak partial observations and instabilities in the XFEL beam position lead to new types of experimental errors. The quality of Bragg intensity estimates deriving from serial crystallography is therefore contingent upon assumptions made while modeling these data. Here it is observed that serial femtosecond crystallography (SFX) Bragg reflections do not follow a unimodal Gaussian distribution and it is recommended that an idealized assumption of single Gaussian peak profiles be relaxed to incorporate apparent asymmetries when processing SFX data. The phenomenon is illustrated by re-analyzing data collected from microcrystals of the Blastochloris viridis photosynthetic reaction center and comparing these intensity observations with conventional synchrotron data. The results show that skewness in the SFX observations captures the essence of the Wilson plot and an empirical treatment is suggested that can help to separate the diffraction Bragg intensity from the background.

  13. Bergström, J.H., Birchenough, G.M., Katona, G., Schroeder, B.O., Schütte, A., Ermund, A., Johansson, M.E., Hansson, G.C. (2016) Gram-positive bacteria are held at a distance in the colon mucus by the lectin-like protein ZG16. PNAS, 113(48), 13833-13838. [FULLTEXT
    The distal colon functions as a bioreactor and harbors an enormous amount of bacteria in a mutualistic relationship with the host. The microbiota have to be kept at a safe distance to prevent inflammation, something that is achieved by a dense inner mucus layer that lines the epithelial cells. The large polymeric nets made up by the heavily O-glycosylated MUC2 mucin forms this physical barrier. Proteomic analyses of mucus have identified the lectin-like protein ZG16 (zymogen granulae protein 16) as an abundant mucus component. To elucidate the function of ZG16, we generated recombinant ZG16 and studied Zg16-/- mice. ZG16 bound to and aggregated Gram-positive bacteria via binding to the bacterial cell wall peptidoglycan. Zg16-/- mice have a distal colon mucus layer with normal thickness, but with bacteria closer to the epithelium. Using distal colon explants mounted in a horizontal perfusion chamber we demonstrated that treatment of bacteria with recombinant ZG16 hindered bacterial penetration into the mucus. The inner colon mucus of Zg16-/- animals had a higher load of Gram-positive bacteria and showed bacteria with higher motility in the mucus close to the host epithelium compared with cohoused littermate Zg16+/+ The more penetrable Zg16-/- mucus allowed Gram-positive bacteria to translocate to systemic tissues. Viable bacteria were found in spleen and were associated with increased abdominal fat pad mass in Zg16-/- animals. The function of ZG16 reveals a mechanism for keeping bacteria further away from the host colon epithelium.

  14. Katona, G., Garcia-Bonete, M.-J. & Lundholm, I.V. (2016) Estimating the difference between structure-factor amplitudes using multivariate Bayesian inference. Acta cryst. Section A, 72(3), 406-11. [FULLTEXT
    In experimental research referencing two or more measurements to one another is a powerful tool to reduce the effect of systematic errors between different sets of measurements. The interesting quantity is usually derived from two measurements on the same sample under different conditions. While an elaborate experimental design is essential for improving the estimate, the data analysis should also maximally exploit the covariance between the measurements. In X-ray crystallography the difference between structure-factor amplitudes carries important information to solve experimental phasing problems or to determine time-dependent structural changes in pump-probe experiments. Here a multivariate Bayesian method was used to analyse intensity measurement pairs to determine their underlying structure-factor amplitudes and their differences. The posterior distribution of the model parameter was approximated with a Markov chain Monte Carlo algorithm. The described merging method is shown to be especially advantageous when systematic and random errors result in recording negative intensity measurements.

  15. Gógl, G., Alexa, A., Kiss, B., Katona, G., Kovács, M., Bodor, A., Reményi, A. & Nyitray, L, (2016) Structural Basis of Ribosomal S6 Kinase 1 (RSK1) Inhibition by S100B Protein: MODULATION OF THE EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK) SIGNALING CASCADE IN A CALCIUM-DEPENDENT WAY. J. Biol. Chem, 291, 11-27. [FULLTEXT
    Mitogen-activated protein kinases (MAPK) promote MAPK-activated protein kinase activation. In the MAPK pathway responsible for cell growth, ERK2 initiates the first phosphorylation event on RSK1, which is inhibited by Ca2+-binding S100 proteins in malignant melanomas. Here, we present a detailed in vitro biochemical and structural characterization of the S100B-RSK1 interaction. The Ca2+-dependent binding of S100B to the calcium/calmodulin-dependent protein kinase (CaMK)-type domain of RSK1 is reminiscent of the better known binding of calmodulin to CaMKII. Although S100B-RSK1 and the calmodulin-CAMKII system are clearly distinct functionally, they demonstrate how unrelated intracellular Ca2+-binding proteins could influence the activity of the CaMK domain-containing protein kinases. Our crystallographic, small angle x-ray scattering, and NMR analysis revealed that S100B forms a “fuzzy” complex with RSK1 peptide ligands. Based on fast-kinetics experiments, we conclude that the binding involves both conformation selection and induced fit steps. Knowledge of the structural basis of this interaction could facilitate therapeutic targeting of melanomas.


  16. Lundholm, I.V., Rodilla, H., Wahlgren, W.Y., Duelli, A., Bourenkov, G., Vukusic, J., Friedman, R., Stake, J., Schneider, T. & Katona, G. (2015) Terahertz radiation induces non-thermal structural changes associated with Fröhlich condensation in a protein crystal. Structural Dynamics, 2(5), 054702 [FULLTEXT
    Whether long-range quantum coherent states could exist in biological systems, and beyond low-temperature regimes where quantum physics is known to be applicable, has been the subject to debate for decades. It was proposed by Fröhlich that vibrational modes within protein molecules can order and condense into a lowest-frequency vibrational mode in a process similar to Bose-Einstein condensation, and thus that macroscopic coherence could potentially be observed in biological systems. Despite the prediction of these so-called Fröhlich condensates almost five decades ago, experimental evidence thereof has been lacking. Here, we present the first experimental observation of Fröhlich condensation in a protein structure. To that end, and to overcome the challenges associated with probing low-frequency molecular vibrations in proteins (which has hampered understanding of their role in proteins' function), we combined terahertz techniques with a highly sensitive X-ray crystallographic method to visualize low-frequency vibrational modes in the protein structure of hen-egg white lysozyme. We found that 0.4 THz electromagnetic radiation induces non-thermal changes in electron density. In particular, we observed a local increase of electron density in a long α-helix motif consistent with a subtle longitudinal compression of the helix. These observed electron density changes occur at a low absorption rate indicating that thermalization of terahertz photons happens on a micro- to milli-second time scale, which is much slower than the expected nanosecond time scale due to damping of delocalized low frequency vibrations. Our analyses show that the micro- to milli-second lifetime of the vibration can only be explained by Fröhlich condensation, a phenomenon predicted almost half a century ago, yet never experimentally confirmed.

  17. Andersson, K.M., Brisslert, M., Cavallini, N.F., Svensson, M.N., Welin, A., Erlandsson, M.C., Ciesielski, M.J., Katona, G. & Bokarewa, M.I. (2015) Survivin co-ordinates formation of follicular T-cells acting in synergy with Bcl-6. Oncotarget, 6(24), 20043-57 [FULLTEXT
    Follicular T helper (Tfh) cells are recognized by the expression of CXCR5 and the transcriptional regulator Bcl-6. Tfh cells control B cell maturation and antibody production, and if deregulated, may lead to autoimmunity. Here, we study the role of the proto-oncogene survivin in the formation of Tfh cells. We show that blood Tfh cells of patients with the autoimmune condition rheumatoid arthritis, have intracellular expression of survivin. Survivin was co-localized with Bcl-6 in the nuclei of CXCR5+CD4 lymphocytes and was immunoprecipitated with the Bcl-6 responsive element of the target genes. Inhibition of survivin in arthritic mice led to the reduction of CXCR5+ Tfh cells and to low production of autoantibodies. Exposure to survivin activated STAT3 and induced enrichment of PD-1+Bcl-6+ subset within Tfh cells. Collectively, our study demonstrates that survivin belongs to the Tfh cell phenotype and ensures their optimal function by regulating transcriptional activity of Bcl-6.

  18. Gráf, L., Molnár, T., Kardos, J., Gáspári, Z. & Katona, G. (2015) The role of structural flexibility and stability in the interaction of serine proteases with their inhibitors. Curr. Protein Pept. Sci., 16(6), 521-31. [FULLTEXT
    Serine proteases and their natural inhibitors have long been served as excellent models for studying (primary, secondary and tertiary) structure - activity relationships of biologically interacting proteins. As protein flexibility has been accepted as a “fourth dimension” of the protein structure, its contribution to the binding process has gained much interest. In this article we review extreme cases of serine protease interactions with canonical serine protease inhibitors that provide unique insights into the dynamics of protein- protein interactions. The major conclusions of our review article are: a) taxon-specific inhibitory effects of two highly homologous protease inhibitors from Schistocerca gregaria (SGCI and SGTI), as investigated by H/D exchange experiments and NMR spectroscopy, are due to their differential flexibilities, b) stabilities of some protease and inhibitor complexes, the wide-spread and increased flexibility of some segments in the protein-protein complexes, as studied by X-ray crystallography and NMR-spectroscopy, appear to be proportional to the physical stability of the complex.

  19. Malmerberg, E., Bovee-Geurts, P.H.M., Katona, G., Deupi, X., Arnlund, D., Wickstrand, C., Johansson, L.C., Westenhoff, S., Nazarenko, E., Schertler, G.F.X., Menzel, A., de Grip, W.J. & Neutze, R. (2015) Conformational activation of visual rhodopsin in native disc membranes. Science Signaling, 8(367), ra26. [FULLTEXT
    Rhodopsin is the G protein–coupled receptor (GPCR) that serves as a dim-light receptor for vision in vertebrates. We probed light-induced conformational changes in rhodopsin in its native membrane environment at room temperature using time-resolved wide-angle x-ray scattering. We observed a rapid conformational transition that is consistent with an outward tilt of the cytoplasmic portion of transmembrane helix 6 concomitant with an inward movement of the cytoplasmic portion of transmembrane helix 5. These movements were considerably larger than those reported from the basis of crystal structures of activated rhodopsin, implying that light activation of rhodopsin involves a more extended conformational change than was previously suggested.

  20. Bodor, A., Radnai, L., Hetényi, C., Rapali, R., Láng, A., Kövér, K. E.,Perczel, A., Wahlgren, W. Y., Katona, G., & Nyitray, L. (2014) DYNLL2 dynein light chain binds to an extended linear motif of myosin 5a tail that has structural plasticity. Biochemistry, 53(45), 7107-22. [FULLTEXT
    LC8 dynein light chains (DYNLL) are conserved homodimeric eukaryotic hub proteins that participate in diverse cellular processes. Among the binding partners of DYNLL2, myosin 5a (myo5a) is a motor protein involved in cargo transport. Here we provide a profound characterization of the DYNLL2 binding motif of myo5a in free and DYNLL2 bound form by using NMR spectroscopy, X-ray crystallography and molecular dynamics simulations. In the free form the DYNLL2 binding region, located in an intrinsically disordered domain of the myo5a tail, has a nascent helical character. The motif becomes structured and folds into a β-strand upon binding to DYNLL2. Despite differences of the myo5a sequence from the consensus binding motif, one peptide is accommodated in each of the parallel DYNLL2 binding grooves, as for all other known partners. Interestingly, while the core motif shows similar interaction pattern in the binding groove as seen in other complexes, the flanking residues make several additional contacts, thereby lengthening the binding motif. The N-terminal extension folds back and partially blocks the free edge of the β-sheet formed by the binding motif itself. The C-terminal extension contacts the dimer interface and interacts with symmetry related residues of the second myo5a peptide. The involvement of flanking residues of the core binding site of myo5a could modify the quaternary structure of the full-length myo5a and affect its biological functions. Our results deepen the knowledge of the diverse partner recognition of DYNLL proteins and provide an example of a Janus-faced linear motif.

  21. Arnlund, D., Johansson, L.C., Wickstrand, C., Barty, A., Williams, G.J., Malmerberg, E., Davidsson, J., Milathianaki, D., DePonte, D.P., Shoeman, R.L., Wang, D., James, D., Katona, G., Westenhoff, S., White, T.A., Aquila, A., Bari, S., Berntsen, P., Bogan, M., van Driel, T.B., Doak, R.B., Kjær, K.S., Frank, M., Fromme, R., Grotjohann, I., Henning, R., Hunter, M.S., Kirian, R.A., Kosheleva, I., Kupitz, C., Liang, M., Martin, A.V., Nielsen, M.M., Messerschmidt, M., Seibert, M.M., Sjöhamn, J., Stellato, F., Weierstall, U., Zatsepin, N.A., Spence, J.C., Fromme, P., Schlichting, I., Boutet, S., Groenhof, G., Chapman, H.N. & Neutze, R. (2014) Visualizing a protein quake with time-resolved X-ray scattering at a free-electron laser. Nat. methods, 11(9), 923-6. [FULLTEXT
    We describe a method to measure ultrafast protein structural changes using time-resolved wide-angle X-ray scattering at an X-ray free-electron laser. We demonstrated this approach using multiphoton excitation of the Blastochloris viridis photosynthetic reaction center, observing an ultrafast global conformational change that arises within picoseconds and precedes the propagation of heat through the protein. This provides direct structural evidence for a 'protein quake': the hypothesis that proteins rapidly dissipate energy through quake-like structural motions.

  22. Lundholm, I., Wahlgren, W. Y.,Piccirilli, F.,Di Pietro, P., Duelli, A.,Berntsson, O., Lupi, S., Perucchi, A. & Katona, G. (2014) Terahertz absorption of illuminated photosynthetic reaction center solution: a signature of photoactivation? RSC Advances, 4, 25502-9. [FULLTEXT
    Photosynthetic reaction centers develop a stable charge separated state upon illumination. To investigate the molecular vibrations associated with the illuminated state of a reaction center we recorded terahertz absorption spectra of the photosynthetic reaction center from Rhodobacter sphaeroides in the dark and upon illumination and observed a small, but significant THz absorption increase in the 20 to 130 cm−1 spectral region. Reaction centers show very similar terahertz absorption increase when solubilized in detergents and in a lipidic sponge phase indicating that the nature of the bulk solvent has limited influence on the vibrational spectrum. The absorption change of the isolated LM subunit is very similar to that of the intact reaction center. Through temperature control experiments we show that 89% of the absorption change is likely attributed to the non-thermal activation of the protein molecules. These results indicate that picosecond molecular vibrations change primarily in the cofactors and/or in the evolutionary conserved core of the reaction center upon illumination, whereas the nuclear motions of the H-subunit and the bulk solvent have limited impact on the terahertz spectral changes.

  23. Duelli, A., Kiss, B., Lundholm, I., Bodor, A., Petoukhov, M.V., Svergun, D.I., Nyitray, L. & Katona G. (2014) The C-Terminal Random Coil Region Tunes the Ca2+-Binding Affinity of S100A4 through Conformational Activation. PLoS One, 9(5), e97654. [PubMed] [FULLTEXT
    S100A4 interacts with many binding partners upon Ca2+ activation and is strongly associated with increased metastasis formation. In order to understand the role of the C-terminal random coil for the protein function we examined how small angle X-ray scattering of the wild-type S100A4 and its C-terminal deletion mutant (residues 1–88, Δ13) changes upon Ca2+ binding. We found that the scattering intensity of wild-type S100A4 changes substantially in the 0.15–0.25 Å−1 q-range whereas a similar change is not visible in the C-terminus deleted mutant. Ensemble optimization SAXS modeling indicates that the entire C-terminus is extended when Ca2+ is bound. Pulsed field gradient NMR measurements provide further support as the hydrodynamic radius in the wild-type protein increases upon Ca2+ binding while the radius of Δ13 mutant does not change. Molecular dynamics simulations provide a rational explanation of the structural transition: the positively charged C-terminal residues associate with the negatively charged residues of the Ca2+-free EF-hands and these interactions loosen up considerably upon Ca2+-binding. As a consequence the Δ13 mutant has increased Ca2+ affinity and is constantly loaded at Ca2+ concentration ranges typically present in cells. The activation of the entire C-terminal random coil may play a role in mediating interaction with selected partner proteins of S100A4.

  24. Johansson, L.C., Arnlund, D., Katona, G., White, T.A., Barty, A., DePonte, D.P., Shoeman, R.L., Wickstrand, C., Sharma, A., Williams, G.J., Aquila, A., Bogan, M.J., Caleman, C., Davidsson, J., Doak, R.B., Frank, M., Fromme, R., Galli, L., Grotjohann, I., Hunter, M.S., Kassemeyer, S., Kirian, R.A., Kupitz, C., Liang, M., Lomb, L., Malmerberg, E., Martin, A.V., Messerschmidt, M., Nass, K., Redecke, L., Seibert, M,M., Sjöhamn, J., Steinbrener, J., Stellato, F., Wang, D., Wahlgren W.Y., Weierstall, U., Westenhoff, S., Zatsepin, N.A., Boutet, S., Spence, J.C., Schlichting, I., Chapman, H.N., Fromme, P. & Neutze, R. (2014) Structure of a photosynthetic reaction centre determined by serial femtosecond crystallography. Nat. Communications, 280(22), 5750-63. [PubMed] [FULLTEXT
    Serial femtosecond crystallography is an X-ray free-electron-laser-based method with considerable potential to have an impact on challenging problems in structural biology. Here we present X-ray diffraction data recorded from microcrystals of the Blastochloris viridis photosynthetic reaction centre to 2.8 Å resolution and determine its serial femtosecond crystallography structure to 3.5 Å resolution. Although every microcrystal is exposed to a dose of 33 MGy, no signs of X-ray-induced radiation damage are visible in this integral membrane protein structure.

  25. Molnar, T., Vörös, J., Szeder, B., Takats, K., Kardos, J., Katona, G. & Graf, L. (2013) Comparison of complexes formed by a crustacean and a vertebrate trypsin with bovine pancreatic trypsin inhibitor - the key to achieving extreme stability? FEBS Journal, 280(22), 5750-63. [PubMed] [FULLTEXT
    This paper provides evidence for the extremely high resistance of a complex of crayfish trypsin (CFT) and bovine pancreatic trypsin inhibitor (BPTI) against heating and chemical denaturing agents such as sodium dodecyl sulfate (SDS) and urea. To dissociate this complex, 15 min boiling in SDS was necessary, compared to a complex of bovine trypsin (BT) (EC and BPTI, which dissociates in SDS without boiling. The CFT–BPTI complex remained stable even in 9 m urea, while the BT–BPTI complex started to dissociate at concentrations of approximately 4 m urea. The melting temperatures of the BT–BPTI and CFT–BPTI complexes, as determined by differential scanning calorimetry, were found to be 79.6 and 100.1 °C, respectively. The behaviour of the apo-enzymes - CFT was found to have a less stable structure compared to BT - did not provide a definite indication regarding the differential effects on their stabilities. To explore the structural features responsible for this extreme stability, we crystallized CFT in complex with BPTI, and identified extended contacts compared to the BT-BPTI complex. Comparison of the B-factors of similar trypsin–trypsin inhibitor complexes suggests that molecular flexibility of the components is also required for the strong protein–protein interaction. Although the structural reason for the extreme stability of the CFT–BPTI complex is not yet fully understood, our study may be a starting point for the development of new protein complexes with enhanced stability.

  26. Redecke, L., Nass, K., Deponte, D. P., White, T. A., Rehders, D., Barty, A., Stellato, F., Liang, M., Barends, T. R., Boutet, S., Williams, G. J., Messerschmidt, M., Seibert, M. M., Aquila, A., Arnlund, D., Bajt, S., Barth, T., Bogan, M. J., Caleman, C., Chao, T. C., Doak, R. B., Fleckenstein, H., Frank, M., Fromme, R., Galli, L., Grotjohann, I., Hunter, M. S., Johansson, L. C., Kassemeyer, S., Katona, G., Kirian, R. A., Koopmann, R., Kupitz, C., Lomb, L., Martin, A. V., Mogk, S., Neutze, R., Shoeman, R. L., Steinbrener, J., Timneanu, N., Wang, D., Weierstall, U., Zatsepin, N. A., Spence, J. C., Fromme, P., Schlichting, I., Duszenko, M., Betzel, C. & Chapman, H. N. (2013) Natively Inhibited Trypanosoma brucei Cathepsin B Structure Determined by Using an X-ray Laser. Science, 339(6116), 227-30. [PubMed] [FULLTEXT
    The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtain the room-temperature 2.1 Å resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the “diffraction before destruction” approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals.

  27. Wahlgren, W.Y., Omran, H., von Stetten, D., Royant, A., van der Post, S. & Katona, G. (2012) Structural Characterization of Bacterioferritin from Blastochloris viridis. PloS One, 7(10),e46992. [PubMed] [FULLTEXT
    Iron storage and elimination of toxic ferrous iron are the responsibility of bacterioferritins in bacterial species. Bacterioferritins are capable of oxidizing iron using molecular oxygen and import iron ions into the large central cavity of the protein, where they are stored in a mineralized form. We isolated, crystallized bacterioferritin from the microaerophilic/anaerobic, purple non-sulfur bacterium Blastochloris viridis and determined its amino acid sequence and X-ray structure. The structure and sequence revealed similarity to other purple bacterial species with substantial differences in the pore regions. Static 3- and 4-fold pores do not allow the passage of iron ions even though structural dynamics may assist the iron gating. On the other hand the B-pore is open to water and larger ions in its native state. In order to study the mechanism of iron import, multiple soaking experiments were performed. Upon Fe(II) and urea treatment the ferroxidase site undergoes reorganization as seen in bacterioferritin from Escherichia coli and Pseudomonas aeruginosa. When soaking with Fe(II) only, a closely bound small molecular ligand is observed close to Fe1 and the coordination of Glu94 to Fe2 changes from bidentate to monodentate. DFT calculations indicate that the bound ligand is most likely a water or a hydroxide molecule representing a product complex. On the other hand the different soaking treatments did not modify the conformation of other pore regions.

  28. Boutet, S., Lomb, L., Williams, G. J., Barends, T. R., Aquila, A., Doak, R. B., Weierstall, U., Deponte, D. P., Steinbrener, J., Shoeman, R. L., Messerschmidt, M., Barty, A., White, T. A., Kassemeyer, S., Kirian, R. A., Seibert, M. M., Montanez, P. A., Kenney, C., Herbst, R., Hart, P., Pines, J., Haller, G., Gruner, S. M., Philipp, H. T., Tate, M. W., Hromalik, M., Koerner, L. J., van Bakel, N., Morse, J., Ghonsalves, W., Arnlund, D., Bogan, M. J., Caleman, C., Fromme, R., Hampton, C. Y., Hunter, M. S., Johansson, L., Katona, G., Kupitz, C., Liang, M., Martin, A. V., Nass, K., Redecke, L., Stellato, F., Timneanu, N., Wang, D., Zatsepin, N. A., Schafer, D., Defever, J., Neutze, R., Fromme, P., Spence, J. C., Chapman, H. N. & Schlichting, I. (2012) High-resolution protein structure determination by serial femtosecond crystallography. Science, 337(6092),362-4. [PubMed] [FULLTEXT
    Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (<1×1×3 μm3) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.

  29. Kiss, B., Duelli, A., Radnai, L., Kekesi, A. K., Katona, G. & Nyitray, L. (2012) Crystal structure of the S100A4–nonmuscle myosin IIA tail fragment complex reveals an asymmetric target binding mechanism. PNAS, 109(16),6048-53. [PubMed] [FULLTEXT
    S100A4 is a member of the S100 family of calcium-binding proteins that is directly involved in tumor metastasis. It binds to the nonmuscle myosin IIA (NMIIA) tail near the assembly competence domain (ACD) promoting filament disassembly, which could be associated with increasing metastatic potential of tumor cells. Here, we investigate the mechanism of S100A4–NMIIA interaction based on binding studies and the crystal structure of S100A4 in complex with a 45-residue-long myosin heavy chain fragment. Interestingly, we also find that S100A4 binds as strongly to a homologous heavy chain fragment of nonmuscle myosin IIC as to NMIIA. The structure of the S100A4–NMIIA complex reveals a unique mode of interaction in the S100 family: A single, predominantly α-helical myosin chain is wrapped around the Ca2+-bound S100A4 dimer occupying both hydrophobic binding pockets. Thermal denaturation experiments of coiled-coil forming NMIIA fragments indicate that the coiled-coil partially unwinds upon S100A4 binding. Based on these results, we propose a model for NMIIA filament disassembly: Part of the random coil tailpiece and the C-terminal residues of the coiled-coil are wrapped around an S100A4 dimer disrupting the ACD and resulting in filament dissociation. The description of the complex will facilitate the design of specific drugs that interfere with the S100A4–NMIIA interaction.

  30. Johansson, L. C., Arnlund, D., White, T. A., Katona, G., Deponte, D. P., Weierstall, U., Doak, R. B., Shoeman, R. L., Lomb, L., Malmerberg, E., Davidsson, J., Nass, K., Liang, M., Andreasson, J., Aquila, A., Bajt, S., Barthelmess, M., Barty, A., Bogan, M. J., Bostedt, C., Bozek, J. D., Caleman, C., Coffee, R., Coppola, N., Ekeberg, T., Epp, S. W., Erk, B., Fleckenstein, H., Foucar, L., Graafsma, H., Gumprecht, L., Hajdu, J., Hampton, C. Y., Hartmann, R., Hartmann, A., Hauser, G., Hirsemann, H., Holl, P., Hunter, M. S., Kassemeyer, S., Kimmel, N., Kirian, R. A., Maia, F. R., Marchesini, S., Martin, A. V., Reich, C., Rolles, D., Rudek, B., Rudenko, A., Schlichting, I., Schulz, J., Seibert, M. M., Sierra, R. G., Soltau, H., Starodub, D., Stellato, F., Stern, S., Struder, L., Timneanu, N., Ullrich, J., Wahlgren, W. Y., Wang, X., Weidenspointner, G., Wunderer, C., Fromme, P., Chapman, H. N., Spence, J. C. & Neutze, R. (2012) Lipidic phase membrane protein serial femtosecond crystallography. Nat. Methods, 9(3), 263 - 5. [PubMed] [FULLTEXT
    X-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. Here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-FEL beam using a sponge phase micro-jet.

  31. Malmerberg, E., Omran, Z., Hub, J. S., Li, X., Katona, G., Westenhoff, S., Johansson, L. C., Andersson, M., Cammarata, M., Wulff, M., Spoel, D., Davidsson, J., Specht, A. & Neutze, R. (2011) Time-Resolved WAXS Reveals Accelerated Conformational Changes in Iodoretinal-Substituted Proteorhodopsin. Biophys. J., 101(6), 1345 - 1353. [PubMed] [FULLTEXT
    Time-resolved wide-angle x-ray scattering (TR-WAXS) is an emerging biophysical method which probes protein conformational changes with time. Here we present a comparative TR-WAXS study of native green-absorbing proteorhodopsin (pR) from SAR86 and a halogenated derivative for which the retinal chromophore has been replaced with 13-desmethyl-13-iodoretinal (13-I-pR). Transient absorption spectroscopy differences show that the 13-I-pR photocycle is both accelerated and displays more complex kinetics than native pR. TR-WAXS difference data also reveal that protein structural changes rise and decay an order-of-magnitude more rapidly for 13-I-pR than native pR. Despite these differences, the amplitude and nature of the observed helical motions are not significantly affected by the substitution of the retinal's C-20 methyl group with an iodine atom. Molecular dynamics simulations indicate that a significant increase in free energy is associated with the 13-cis conformation of 13-I-pR, consistent with our observation that the transient 13-I-pR conformational state is reached more rapidly. We conclude that although the conformational trajectory is accelerated, the major transient conformation of pR is unaffected by the substitution of an iodinated retinal chromophore.

  32. Rapali, P., Radnai, L., Süveges, D., Harmat, V., Tölgyesi, F., Wahlgren, W.Y., Katona, G., Nyitray, L. & Pál, G. (2011) Directed Evolution Reveals the Binding Motif Preference of the LC8/DYNLL Hub Protein and Predicts Large Numbers of Novel Binders in the Human Proteome. PloS One, 6(4), e18818. [PubMed] [FULLTEXT
    LC8 dynein light chain (DYNLL) is a eukaryotic hub protein that is thought to function as a dimerization engine. Its interacting partners are involved in a wide range of cellular functions. In its dozens of hitherto identified binding partners DYNLL binds to a linear peptide segment. The known segments define a loosely characterized binding motif: [D/S](-4)K(-3)X(-2)[T/V/I](-1)Q(0)[T/V](1)[D/E](2). The motifs are localized in disordered segments of the DYNLL-binding proteins and are often flanked by coiled coil or other potential dimerization domains. Based on a directed evolution approach, here we provide the first quantitative characterization of the binding preference of the DYNLL binding site. We displayed on M13 phage a naïve peptide library with seven fully randomized positions around a fixed, naturally conserved glutamine. The peptides were presented in a bivalent manner fused to a leucine zipper mimicking the natural dimer to dimer binding stoichiometry of DYNLL-partner complexes. The phage-selected consensus sequence V(-5)S(-4)R(-3)G(-2)T(-1)Q(0)T(1)E(2) resembles the natural one, but is extended by an additional N-terminal valine, which increases the affinity of the monomeric peptide twentyfold. Leu-zipper dimerization increases the affinity into the subnanomolar range. By comparing crystal structures of an SRGTQTE-DYNLL and a dimeric VSRGTQTE-DYNLL complex we find that the affinity enhancing valine is accommodated in a binding pocket on DYNLL. Based on the in vitro evolved sequence pattern we predict a large number of novel DYNLL binding partners in the human proteome. Among these EML3, a microtubule-binding protein involved in mitosis contains an exact match of the phage-evolved consensus and binds to DYNLL with nanomolar affinity. These results significantly widen the scope of the human interactome around DYNLL and will certainly shed more light on the biological functions and organizing role of DYNLL in the human and other eukaryotic interactomes.

  33. Wahlgren, W.Y., Pál, G., Kardos, J., Porrogi, P., Szenthe, B., Patthy, A., Gráf, L. & Katona, G. (2011) The catalytic aspartate is protonated in the Michaelis complex formed between trypsin and an in vitro evolved substrate-like inhibitor: a refined mechanism of serine protease action. J. Biol. Chem., 286(5), 3587-96. [PubMed] [FULLTEXT] [MOVIE]  
    The mechanism of serine proteases prominently illustrates how charged amino acid residues and proton transfer events facilitate enzyme catalysis. Here we present an ultrahigh resolution (0.93 A) X-ray structure of a complex formed between trypsin and a canonical inhibitor acting through a substrate-like mechanism. The electron density indicates the protonation state of all catalytic residues where the catalytic histidine is, as expected, in its neutral state prior to the acylation step by the catalytic serine. The carboxyl group of the catalytic aspartate displays an asymmetric electron density so that Od2-Cg bond appears to be a double bond, with Od2 involved in a hydrogen bond to His-57 and Ser-214. Only when Asp-102 is protonated on Od1 atom a DFT simulation could reproduce the observed electron density. The presence of a putative hydrogen atom is also confirmed by a residual mFobs-DFcalc density above 2.5 sigma next to Od1. As a possible functional role for the neutral aspartate in the active site, we propose that in the substrate bound form the neutral aspartate residue helps to keep the pKa of the histidine sufficiently low, in the active neutral form. When the histidine receives a proton during the catalytic cycle, the aspartate becomes simultaneously negatively charged providing additional stabilization for the protonated histidine and indirectly to the tetrahedral intermediate. This novel proposal unifies the seemingly conflicting experimental observations, which were previously seen as either supporting the charge relay mechanism or the neutral-pKa histidine theory.

  34. 2006-2010

  35. Westenhoff, S., Malmerberg, E., Arnlund, D., Johansson, L., Nazarenko, E., Cammarata, M., Davidsson, J., Chaptal, V., Abramson, J., Katona, G., Menzel, A. & Neutze, R. (2010) Rapid readout detector captures protein time-resolved WAXS. Nat. Methods, 7(10), 775-6. [PubMed] [FULLTEXT
    No Abstract.

  36. Wöhri, A. B., Katona, G., Johansson, L. C., Fritz, E., Malmerberg, E., Andersson, M., Vincent, J., Eklund, M., Cammarata, M., Wulff, M., Davidsson, J., Groenhof, G. & Neutze, R. (2010) Light-Induced Structural Changes in a Photosynthetic Reaction Center Caught by Laue Diffraction Science, 328, 630-633. [PubMed] [FULLTEXT
    Photosynthetic reaction centers convert the energy content of light into a transmembrane potential difference and so provide the major pathway for energy input into the biosphere. We applied time-resolved Laue diffraction to study light-induced conformational changes in the photosynthetic reaction center complex of Blastochloris viridis. The side chain of TyrL162, which lies adjacent to the special pair of bacteriochlorophyll molecules that are photooxidized in the primary light conversion event of photosynthesis, was observed to move 1.3 angstroms closer to the special pair after photoactivation. Free energy calculations suggest that this movement results from the deprotonation of this conserved tyrosine residue and provides a mechanism for stabilizing the primary charge separation reactions of photosynthesis.

  37. Westenhoff, S., Nazarenko, E., Malmerberg, E., Davidsson, J., Katona, G. & Neutze, R. (2010) Time-resolved structural studies of protein reaction dynamics: a smorgasbord of X-ray approaches. Acta Cryst.  A66, 207–219. [FULLTEXT] 
    Proteins undergo conformational changes during their biological function. As such, a high-resolution structure of a protein's resting conformation provides a starting point for elucidating its reaction mechanism, but provides no direct information concerning the protein's conformational dynamics. Several X-ray methods have been developed to elucidate those conformational changes that occur during a protein's reaction, including time-resolved Laue diffraction and intermediate trapping studies on three-dimensional protein crystals, and time-resolved wide-angle X-ray scattering and X-ray absorption studies on proteins in the solution phase. This review emphasizes the scope and limitations of these complementary experimental approaches when seeking to understand protein conformational dynamics. These methods are illustrated using a limited set of examples including myoglobin and haemoglobin in complex with carbon monoxide, the simple light-driven proton pump bacteriorhodopsin, and the superoxide scavenger superoxide reductase. In conclusion, likely future developments of these methods at synchrotron X-ray sources and the potential impact of emerging X-ray free-electron laser facilities are speculated upon.

  38. Johansson, L. C., Wöhri, A.B., Katona, G., Engström, S. & Neutze, R. (2009) Membrane protein crystallization from lipidic phases. Curr. Opin. Struct. Biol., 19(4), 372-8. [PubMed] [FULLTEXT] 
    Membrane protein structural biology is enjoying a steady acceleration in the rate of success. Nevertheless, numerous membrane protein targets are resistant to the traditional approach of directly crystallizing detergent solubilized and purified protein and the 'niche market' of lipidic phase crystallization is emerging as a powerful complement. These approaches, including lipidic cubic phase, lipidic sponge phase, and bicelle crystallization methods, all immerse purified membrane protein within a lipid rich matrix before crystallization. This environment is hypothesized to contribute to the protein's long-term structural stability and thereby favor crystallization. Spectacular recent successes include the high-resolution structures of the beta(2)-adrenergic G-protein-coupled receptor, the A(2A) adenosine G-protein-coupled receptor, and the mitochondrial voltage dependent anion channel. In combination with technical innovations aiming to popularize these methods, lipidic phase crystallization approaches can be expected to deliver an increasing scientific impact as the field develops.

  39. Wöhri, A. B., Wahlgren W. Y., Malmerberg, E., Johansson, L.C., Neutze, R. & Katona, G. (2009) Lipidic sponge phase crystal structure of a photosynthetic reaction center reveals peripheral lipids. Biochemistry, 48(41), 9831-8. [PubMed] [FULLTEXT
    Membrane proteins are embedded in a lipid bilayer and maintain strong interactions with lipid molecules. Tightly bound lipids are responsible for vertical positioning and integration of proteins in the membrane and for assembly of multisubunit complexes and occasionally act as substrates. In this work we present the lipidic sponge phase crystal structure of the reaction center from Blastochloris viridis to 1.86 A, which reveals lipid molecules interacting with the protein surface. A diacylglycerol molecule is bound, through a thioether bond, to the N-terminus of the tetraheme cytochrome c subunit. From the electron density recovered at the Q(B) site and the observed change in recombination kinetics in lipidic sponge phase-grown crystals, the mobile ubiquinone appears to be displaced by a monoolein molecule. A 36 A long electron density feature is observed at the interface of transmembrane helices belonging to the H- and M-subunits, probably arising from an unidentified lipid.

  40. Andersson, M., Malmerberg, E., Westenhoff, S., Katona, G., Cammarata, M., Wöhri, A. B., Johansson, L. C., Ewald, F., Eklund, M., Wulff, M., Davidsson, J. & Neutze, R. (2009) Structural dynamics of light driven proton pumps. Structure, 17(9), 1265-75. [PubMed] [FULLTEXT
    Bacteriorhodopsin and proteorhodopsin are simple heptahelical proton pumps containing a retinal chromophore covalently bound to helix G via a protonated Schiff base. Following the absorption of a photon, all-trans retinal is isomerized to a 13-cis conformation, initiating a sequence of conformational changes driving vectorial proton transport. In this study we apply time-resolved wide-angle X-ray scattering to visualize in real time the helical motions associated with proton pumping by bacteriorhodopsin and proteorhodopsin. Our results establish that three conformational states are required to describe their photocycles. Significant motions of the cytoplasmic half of helix F and the extracellular half of helix C are observed prior to the primary proton transfer event, which increase in amplitude following proton transfer. These results both simplify the structural description to emerge from intermediate trapping studies of bacteriorhodopsin and reveal shared dynamical principles for proton pumping.

  41. Wöhri, A., Johansson, L., Wadsten, P. H., Wahlgren, W. Y., Fischer, G., Horsefield, R., Katona, G., Nyblom, M., Oberg, F., Young, G., Cogdell, R. J., Fraser, N. J., Engström, S. & Neutze, R. (2008) A Lipidic-Sponge Phase Screen for Membrane Protein Crystallization.  Structure, 16, 1-7.[PubMed] [FULLTEXT] 
    A major current deficit in structural biology is the lack of high-resolution structures of eukaryotic membrane proteins, many of which are key drug targets for the treatment of disease. Numerous eukaryotic membrane proteins require specific lipids for their stability and activity, and efforts to crystallize and solve the structures of membrane proteins that do not address the issue of lipids frequently end in failure rather than success. To help address this problem, we have developed a sparse matrix crystallization screen consisting of 48 lipidic-sponge phase conditions. Sponge phases form liquid lipid bilayer environments which are suitable for conventional hanging- and sitting-drop crystallization experiments. Using the sponge phase screen, we obtained crystals of several different membrane proteins from bacterial and eukaryotic sources. We also demonstrate how the screen may be manipulated by incorporating specific lipids such as cholesterol; this modification led to crystals being recovered from a bacterial photosynthetic core complex.

  42. Burgess, S. G., Messiha, H. L., Katona, G., Rigby, S. E. J., Leys, D. & Scrutton, N. S. (2008) Probing the dynamic interface between trimethylamine dehydrogenase (TMADH) and electron transferring flavoprotein (ETF) in the TMADH—2ETF complex: role of the Arg-a237 (ETF) and Tyr-442 (TMADH) residue pair. Biochemistry, 47 (18), 5168–5181. [PubMed] [FULLTEXT] 
    We have used multiple solution state techniques and crystallographic analysis to investigate the importance of a putative transient interaction formed between Arg-alpha237 in electron transferring flavoprotein (ETF) and Tyr-442 in trimethylamine dehydrogenase (TMADH) in complex assembly, electron transfer, and structural imprinting of ETF by TMADH. We have isolated four mutant forms of ETF altered in the identity of the residue at position 237 (alphaR237A, alphaR237K, alphaR237C, and alphaR237E) and with each form studied electron transfer from TMADH to ETF, investigated the reduction potentials of the bound ETF cofactor, and analyzed complex formation. We show that mutation of Arg-alpha237 substantially destabilizes the semiquinone couple of the bound FAD and impedes electron transfer from TMADH to ETF. Crystallographic structures of the mutant ETF proteins indicate that mutation does not perturb the overall structure of ETF, but leads to disruption of an electrostatic network at an ETF domain boundary that likely affects the dynamic properties of ETF in the crystal and in solution. We show that Arg-alpha237 is required for TMADH to structurally imprint the as-purified semiquinone form of wild-type ETF and that the ability of TMADH to facilitate this structural reorganization is lost following (i) redox cycling of ETF, or simple conversion to the oxidized form, and (ii) mutagenesis of Arg-alpha237. We discuss this result in light of recent apparent conflict in the literature relating to the structural imprinting of wild-type ETF. Our studies support a mechanism of electron transfer by conformational sampling as advanced from our previous analysis of the crystal structure of the TMADH-2ETF complex [Leys, D. , Basran, J. , Sutcliffe, M. J., and Scrutton, N. S. (2003) Nature Struct. Biol. 10, 219-225] and point to a key role for the Tyr-442 (TMADH) and Arg-alpha237 (ETF) residue pair in transiently stabilizing productive electron transfer configurations. Our work also points to the importance of Arg-alpha237 in controlling the thermodynamics of electron transfer, the dynamics of ETF, and the protection of reducing equivalents following disassembly of the TMADH-2ETF complex.

  43. Jelinek, B., Katona, G., Fodor, K., Venekei, I., & Gráf L. (2008) The Crystal Structure of a Trypsin-like Mutant Chymotrypsin: The Role of Position 226 in the Activity and Specificity of S189D Chymotrypsin. Protein Journal, 27(2), 79-87. [PubMed] 
    The crystal structure of the S189D+A226G rat chymotrypsin-B mutant has been determined at 2.2 angstroms resolution. This mutant is the most trypsin-like mutant so far in the line of chymotrypsin-to-trypsin conversions, aiming for a more complete understanding of the structural basis of substrate specificity in pancreatic serine proteases. A226G caused significant rearrangements relative to S189D chymotrypsin, allowing an internal conformation of Asp189 which is close to that in trypsin. Serious distortions remain, however, in the activation domain, including zymogen-like features. The pH-profile of activity suggests that the conformation of the S1-site of the mutant is influenced also by the P1 residue of the substrate.

  44. Katona, G., Carpentier, P., Niviere, V., Amara, P., Adam, V., Ohana, J., Tsanov, N. & Bourgeois, D. (2007) Raman-assisted crystallography reveals end-on peroxide intermediates in a non-heme iron enzyme. Science, 316(5823), 449-53.  [PubMed] [FULLTEXT] [MOVIE] 
    Iron-pero Academie_medal2_smallxide intermediates are central in the reaction cycle of many iron-containing biomolecules. We trapped iron(III)-(hydro)peroxo species in crystals of superoxide reductase (SOR), a nonheme mononuclear iron enzyme that scavenges superoxide radicals. X-ray diffraction data at 1.95 angstrom resolution and Raman spectra recorded in crystallo revealed iron-(hydro)peroxo intermediates with the (hydro)peroxo group bound end-on. The dynamic SOR active site promotes the formation of transient hydrogen bond networks, which presumably assist the cleavage of the iron-oxygen bond in order to release the reaction product, hydrogen peroxide.

  45. Wadsten, P., Wohri, A.B., Snijder, A., Katona, G., Gardiner, A.T., Cogdell, R.J., Neutze, R. & Engstrom, S. (2006) Lipidic sponge phase crystallization of membrane proteins. J. Mol. Biol., 364(1), 44-53. [PubMed] [FULLTEXT] 
    Bicontinuous lipidic cubic phases can be used as a host for growing crystals of membrane proteins. Since the cubic phase is stiff, handling is difficult and time-consuming. Moreover, the conventional cubic phase may interfere with the hydrophilic domains of membrane proteins due to the limited size of the aqueous pores. Here, we introduce a new crystallization method that makes use of a liquid analogue of the cubic phase, the sponge phase. This phase facilitates a considerable increase in the allowed size of aqueous domains of membrane proteins, and is easily generalised to a conventional vapour diffusion crystallisation experiment, including the use of nanoliter drop crystallization robots. The appearance of the sponge phase was confirmed by visual inspection, small-angle X-ray scattering and NMR spectroscopy. Crystals of the reaction centre from Rhodobacter sphaeroides were obtained by a conventional hanging-drop experiment, were harvested directly without the addition of lipase or cryoprotectant, and the structure was refined to 2.2 Angstroms resolution. In contrast to our earlier lipidic cubic phase reaction centre structure, the mobile ubiquinone could be built and refined. The practical advantages of the sponge phase make it a potent tool for crystallization of membrane proteins.

  46. Fodor, K., Harmat, V., Neutze, R., Szilagyi, L. Graf, L. & Katona, G. (2006) Enzyme:substrate hydrogen bond shortening during the acylation phase of serine protease catalysis. Biochemistry, 45(7), 2114-21. [PubMed] [FULLTEXT] 
    Atomic resolution (<or=1.2 A) serine protease intermediate structures revealed that the strength of the hydrogen bonds between the enzyme and the substrate changed during catalysis. The well-conserved hydrogen bonds of antiparallel beta-sheet between the enzyme and the substrate become significantly shorter in the transition from a Michaelis complex analogue (Pontastacus leptodactylus (narrow-fingered crayfish) trypsin (CFT) in complex with Schistocerca gregaria (desert locust) trypsin inhibitor (SGTI) at 1.2 A resolution) to an acyl-enzyme intermediate (N-acetyl-Asn-Pro-Ile acyl-enzyme intermediate of porcine pancreatic elastase at 0.95 A resolution) presumably synchronously with the nucleophilic attack on the carbonyl carbon atom of the scissile peptide bond. This is interpreted as an active mechanism that utilizes the energy released from the stronger hydrogen bonds to overcome the energetic barrier of the nucleophilic attack by the hydroxyl group of the catalytic serine. In the CFT:SGTI complex this hydrogen bond shortening may be hindered by the 27I-32I disulfide bridge and Asn-15I of SGTI. The position of the catalytic histidine changes slightly as it adapts to the different nucleophilic attacker during the transition from the Michaelis complex to the acyl-enzyme state, and simultaneously its interaction with Asp-102 and Ser-214 becomes stronger. The oxyanion hole hydrogen bonds provide additional stabilization for acyl-ester bond in the acyl-enzyme than for scissile peptide bond of the Michaelis complex. Significant deviation from planarity is not observed in the reactive bonds of either the Michaelis complex or the acyl-enzyme. In the Michaelis complex the electron distribution of the carbonyl bond is distorted toward the oxygen atom compared to other peptide bonds in the structure, which indicates the polarization effect of the oxyanion hole.

  47. 2001-2005

  48. Katona, G., Andreasson, U., Gourdon, P., Snijder, A., Hansson, Ö., Andreasson, L.-E. & Neutze, R. (2005) Conformational regulation of charge recombination reactions in a photosynthetic bacterial reaction center. Nat. Struct. and Mol. Biol., 12(7), 630-1. [PubMed] [FULLTEXT] 
    Well-ordered crystals of the bacterial photosynthetic reaction centre from Rhodobacter sphaeroides were grown from a lipidic cubic phase. Here, we report the type I crystal packing that results from this crystallisation medium, for which 3D crystals grow as stacked 2D crystals, and the reaction centre X-ray structure is refined to 2.35A resolution. In this crystal form, the location of the membrane bilayer could be assigned with confidence. A cardiolipin-binding site is found at the protein-protein interface within the membrane-spanning region, shedding light on the formation of crystal contacts within the membrane. A chloride-binding site was identified in the membrane-spanning region, which suggests a putative site for interaction with the light-harvesting complex I, the cytochrome bc(1) complex or PufX. Comparisons with the X-ray structures of this reaction centre deriving from detergent-based crystals are drawn, indicating that a slight compression occurs in this lipid-rich environment.

  49. Davidsson J., Poulsen J., Cammarata M., Georgiou P., Wouts R., Katona G., Jacobson, F., Plech, A., Wulff, M., Nyman, G. & Neutze R. (2005) Structural determination of a transient isomer of CH2I2 by picosecond X-ray diffraction. Phys. Rev. Lett., 94, 245503. [FULLTEXT
    Ultrafast time-resolved spectroscopic studies of complex chemical reactions in solution are frequently hindered by difficulties in recovering accurate structural models for transient photochemical species. Time-resolved x-ray and electron diffraction have recently emerged as techniques for probing the structural dynamics of short lived photointermediates. Here we determine the structure of a transient isomer of photoexcited CH2 I2 in solution and observe the downstream reactions of the initial photo-products. Our results illustrate how geminate recombination proceeds via the formation of a transient covalent bond onto the iodine atom remaining with the parent molecule. Further intramolecular rearrangements are thus required for the CH2 I-I isomer to return to CH2 I2 . The generation of I3 from  those iodine radicals escaping the solvent cage is also followed with time.

  50. Fodor K., Harmat V., Hetenyi C., Kardos, J., Antal J., Perczel A., Patthy A., Katona G. & Graf L. (2005) Extended intermolecular interactions in a serine protease-canonical inhibitor complex account for strong and highly specific inhibition. J. Mol. Biol., 350(1), 156-69. [PubMed] [FULLTEXT
    We have previously shown that a trypsin inhibitor from desert locust Schistocerca gregaria (SGTI) is a taxon-specific inhibitor that inhibits arthropod trypsins, such as crayfish trypsin, five orders of magnitude more effectively than mammalian trypsins. Thermal denaturation experiments, presented here, confirm the inhibition kinetics studies; upon addition of SGTI the melting temperatures of crayfish and bovine trypsins increased 27 degrees C and 4.5 degrees C, respectively. To explore the structural features responsible for this taxon specificity we crystallized natural crayfish trypsin in complex with chemically synthesized SGTI. This is the first X-ray structure of an arthropod trypsin and also the highest resolution (1.2A) structure of a trypsin-protein inhibitor complex reported so far. Structural data show that in addition to the primary binding loop, residues P3-P3' of SGTI, the interactions between SGTI and the crayfish enzyme are also extended over the P12-P4 and P4'-P5' regions. This is partly due to a structural change of region P10-P4 in the SGTI structure induced by binding of the inhibitor to crayfish trypsin. The comparison of SGTI-crayfish trypsin and SGTI-bovine trypsin complexes by structure-based calculations revealed a significant interaction energy surplus for the SGTI-crayfish trypsin complex distributed over the entire binding region. The new regions that account for stronger and more specific binding of SGTI to crayfish than to bovine trypsin offer new inhibitor sites to engineer in order to develop efficient and specific protease inhibitors for practical use.

  51. Kénesi, E., Katona, G. & Szilágyi, L. (2003) Structural and evolutionary consequences of unpaired cysteines in trypsinogen. Biochem. Biophys. Res. Comm., 309(4), 749-754. [PubMed] [FULLTEXT

    Vertebrate trypsins usually contain six disulfide bonds but human trypsin 1 (PRSS1) contains only five and human trypsin 2 (PRSS2) contains only four. To elucidate possible evolutionary pathways leading to the loss of disulfide bonds, we have constructed mutants lacking one or two cysteines of four disulfide bonds (C22-C157, C127-C232, C136-C201, and C191-C220) in rat anionic trypsinogen and followed their expression in the periplasm of Escherichia coli. When both cysteines of any of the above-mentioned disulfide bonds were replaced by alanines we found, as expected, proteolytically active enzymes. In the case of C127-C232 (missing from both human trypsins) and C191-C220 both single mutants gave active enzymes although their yield was significantly reduced. In contrast, only one of the single mutants of disulfide bonds C22-C157 and C136-C201 (missing from human trypsin 2) was expressed in E. coli. In the case of these disulfide bonds, we obtained no expression when the solvent accessible molecular surface of the free cysteine residue was the smaller one, indicating that a buried unpaired cysteine was more deleterious than one on the surface of the molecule.

  52. Katona, G., Andreasson, U., Landau, E., Andreasson, L.-E. & Neutze, R. (2003) Lipidic cubic phase crystal structure of the photosynthetic reaction centre from Rhodobacter sphaeroides at 2.35 Ã… resolution. J. Mol. Biol., 331(3), 681-92. [PubMed] [FULLTEXT
    Well-ordered crystals of the bacterial photosynthetic reaction centre from Rhodobacter sphaeroides were grown from a lipidic cubic phase. Here, we report the type I crystal packing that results from this crystallisation medium, for which 3D crystals grow as stacked 2D crystals, and the reaction centre X-ray structure is refined to 2.35A resolution. In this crystal form, the location of the membrane bilayer could be assigned with confidence. A cardiolipin-binding site is found at the protein-protein interface within the membrane-spanning region, shedding light on the formation of crystal contacts within the membrane. A chloride-binding site was identified in the membrane-spanning region, which suggests a putative site for interaction with the light-harvesting complex I, the cytochrome bc(1) complex or PufX. Comparisons with the X-ray structures of this reaction centre deriving from detergent-based crystals are drawn, indicating that a slight compression occurs in this lipid-rich environment.

  53. Katona, G., Wilmouth, R. C., Wright, P. A., Berglund, G. I., Hajdu, J., Neutze, R. & Schofield, C. J. (2002) X-ray structure of a serine protease acyl-enzyme complex at 0.95 A resolution. J. Biol. Chem., 277(24), 21962-21970. [PubMed] [FULLTEXT
    Kinetic analyses led to the discovery that N-acetylated tripeptides with polar residues at P3 are inhibitors of porcine pancreatic elastase (PPE) that form unusually stable acyl-enzyme complexes. Peptides terminating in a C-terminal carboxylate were more potent than those terminating in a C-terminal amide, suggesting recognition by the oxy-anion hole is important in binding. X-ray diffraction data were recorded to 0.95-A resolution for an acyl-enzyme complex formed between PPE and N-acetyl-Asn-Pro-Ile-CO2H at approximately pH 5. The accuracy of the crystallographic coordinates allows structural issues concerning the mechanism of serine proteases to be addressed. Significantly, the ester bond of the acyl-enzyme showed a high level of planarity, suggesting geometric strain of the ester link is not important during catalysis. Several hydrogen atoms could be clearly identified and were included within the model. In keeping with a recent x-ray structure of subtilisin at 0.78 A (1), limited electron density is visible consistent with the putative location of a hydrogen atom approximately equidistant between the histidine and aspartate residues of the catalytic triad. Comparison of this high resolution crystal structure of the acyl-enzyme complex with that of native elastase at 1.1 A (2) showed that binding of the N-terminal part of the substrate can be accommodated with negligible structural rearrangements. In contrast, comparison with structures obtained as part of "time-resolved" studies on the reacting acyl-enzyme complex at >pH 7 (3) indicate small but significant structural differences, consistent with the proposed synchronization of ester hydrolysis and substrate release.

  54. Synnergren, O., Harbst, M., Missalla, T., Larsson, J., Katona, G., Neutze, R. & Wouts, R. (2002) Projecting picosecond lattice dynamics through x-ray topography. Appl. Phys. Lett., 80(20), 3727-3729. [FULLTEXT
    A method for time-resolved x-ray diffraction studies has been demonstrated. As a test case, coherent acoustic phonon propagation into crystalline InSb is observed using a laser plasma x-ray source. An extended x-ray topogram of the semiconductor’s surface was projected onto a high spatial resolution x-ray detector and acoustic phonons were excited by rapidly heating the crystal’s surface with a femtosecond laser pulse. A correlation between the spatial position on the x-ray detector and the time of arrival of the laser pulse was encoded into the experimental geometry by tilting the incident laser pulse with an optical grating. This approach enabled a temporal window of 200 ps to be sampled in a single topogram, thereby negating the disadvantages of pulse-to-pulse fluctuations in
    the intensity and spectrum of the laser-plasma source.

  55. Katona, G., Berglund, G. I., Hajdu, J., Gráf, L. & Szilágyi, L. (2002) Crystal structure reveals basis for the inhibitor resistance of human brain trypsin. J. Mol. Biol., 315(5), 1209-1218. [PubMed] [FULLTEXT
    Severe neurodegradative brain diseases, like Alzheimer, are tightly linked with proteolytic activity in the human brain. Proteinases expressed in the brain, such as human trypsin IV, are likely to be involved in the pathomechanism of these diseases. The observation of amyloid formed in the brain of transgenic mice expressing human trypsin IV supports this hypothesis. Human trypsin IV is also resistant towards all studied naturally occurring polypeptide inhibitors. It has been postulated that the substitution of Gly193 to arginine is responsible for this inhibitor resistance. Here we report the X-ray structure of human trypsin IV in complex with the inhibitor benzamidine at 1.7 A resolution. The overall fold of human trypsin IV is similar to human trypsin I, with a root-mean square deviation of only 0.5 A for all C(alpha) positions. The crystal structure reveals the orientation of the side-chain of Arg193, which occupies an extended conformation and fills the S2' subsite. An analysis of surface electrostatic potentials shows an unusually strong clustering of positive charges around the primary specificity pocket, to which the side-chain of Arg193 also contributes. These unique features of the crystal structure provide a structural basis for the enhanced inhibitor resistance, and enhanced substrate restriction, of human trypsin IV.

  56. Szilágyi, L., Kenesi, E., Katona, G., Kaslik, G., Juhász, G. & Gráf, L. (2001) Comparative in vitro studies on native and recombinant human cationic trypsins. cathepsin B is a possible pathological activator of trypsinogen in pancreatitis. J. Biol. Chem., 276(27), 24574-80. [PubMed] [FULLTEXT] 
    Hereditary pancreatitis, an autosomal dominant disease is believed to be caused by mutation in the human trypsinogen gene. The role of mutations has been investigated by in vitro studies using recombinant rat and human trypsinogen (TG). In this study we compare the enzymatic properties and inhibition by human pancreatic secretory trypsin inhibitor (hPSTI) of the native, postsynthetically modified and recombinant cationic trypsin, and found these values practically identical. We also determined the autolytic stability of recombinant wild type (Hu1Asn21) and pancreatitis-associated (Hu1Ile21) trypsin. Both forms were equally stable. Similarly, we found no difference in the rate of activation of the two zymogens by human cationic and anionic trypsin. Mesotrypsin did not activate either form. The rate of autocatalytic activation of Hu1Asn21 TG and Hu1Ile21 TG was also identical at pH 8 both in the presence and absence of Ca2+. At pH 5 Hu1Ile21 TG autoactivated about twice as fast as Hu1Asn21 TG. The presence of physiological amount of hPSTI completely prevented autoactivation of both zymogens at pH 8 and at pH 5 as well. Cathepsin B readily activated both zymogens although Hu1Ile21 TG was activated about 2.5-3 times as fast as Hu1Asn21 TG. The presence of hPSTI did not prevent the activation of zymogens by cathepsin B. Our results underlie the central role of cathepsin B in the development of different forms of pancreatitis.

Popular-scientific articles

  1. Bourgeois, D., de Rosny, E. & Katona, G. (2007) La cristallographie cinetique : un outil pour filmer les proteines au travail. Biofutur, 280, 48-51. [FULLTEXT]

Books and book chapters

  1. Bourgeois, D., Katona, G., de Rosny, E. & Carpentier, P. (2009) Raman-assisted X-ray crystallography for analysis of biomolecules.  Methods in Molecular Biology, 544, 253-67. [PubMed] [FULLTEXT]


Gergely Katona on ResearchGate