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1.  The C-Terminal Random Coil Region Tunes the Ca2+-Binding Affinity of S100A4 through Conformational Activation 
PLoS ONE  2014;9(5):e97654.
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.
PMCID: PMC4022583  PMID: 24830809
2.  High-Resolution Protein Structure Determination by Serial Femtosecond Crystallography 
Science (New York, N.Y.)  2012;337(6092):362-364.
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 (less than 1 micrometer by 1 micrometer by 3 micrometers) 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.
PMCID: PMC3788707  PMID: 22653729
3.  Natively Inhibited Trypanosoma brucei Cathepsin B Structure Determined by Using an X-ray Laser 
Science (New York, N.Y.)  2012;339(6116):227-230.
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 obtained the room-temperature 2.1 angstrom 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.
PMCID: PMC3786669  PMID: 23196907
4.  Release probability of hippocampal glutamatergic terminals scales with the size of the active zone 
Nature neuroscience  2012;15(7):988-997.
Cortical synapses display remarkable structural, molecular and functional heterogeneity. Our knowledge regarding the relationship between the ultrastructural and functional parameters is still fragmented. Here we asked how the release probability and presynaptic [Ca2+] transients relate to the ultrastructure of rat hippocampal glutamatergic axon terminals. Two-photon Ca2+ imaging-derived optical quantal analysis and correlated electron microscopic reconstructions revealed a tight correlation between the release probability and the active zone area. The peak amplitude of [Ca2+] transients in single boutons also positively correlated with the active zone area. Freeze-fracture immunogold labeling revealed that the voltage-gated Ca2+ channel subunit Cav2.1 and the presynaptic protein Rim1/2 are confined to the active zone and their numbers scale linearly with the active zone area. Gold particles for Cav2.1 showed a nonrandom distribution within the active zones. Our results demonstrate that the number of several active zone proteins, including presynaptic Ca2+ channels, docked vesicles and the release probability scales linearly with the active zone area.
PMCID: PMC3386897  PMID: 22683683
5.  Structural Characterization of Bacterioferritin from Blastochloris viridis 
PLoS ONE  2012;7(10):e46992.
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.
PMCID: PMC3467274  PMID: 23056552
6.  Lipidic phase membrane protein serial femtosecond crystallography 
Nature methods  2012;9(3):263-265.
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.
PMCID: PMC3438231  PMID: 22286383
7.  The Catalytic Aspartate Is Protonated in the Michaelis Complex Formed between Trypsin and an in Vitro Evolved Substrate-like Inhibitor 
The Journal of Biological Chemistry  2010;286(5):3587-3596.
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 Å) 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 the Oδ2–Cγ bond appears to be a double bond, with Oδ2 involved in a hydrogen bond to His-57 and Ser-214. Only when Asp-102 is protonated on Oδ1 atom could a density functional theory simulation reproduce the observed electron density. The presence of a putative hydrogen atom is also confirmed by a residual mFobs − DFcalc density above 2.5 σ next to Oδ1. 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.
PMCID: PMC3030363  PMID: 21097875
Enzyme Catalysis; Enzyme Kinetics; Enzyme Mechanisms; Enzyme Structure; Enzymes; Protease; Protease Inhibitor; Proteolytic Enzymes; Serine Protease; X-ray Crystallography
8.  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  2011;6(4):e18818.
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]-4K-3X-2[T/V/I]-1Q0[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-5S-4R-3G-2T-1Q0T1E2 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.
PMCID: PMC3078936  PMID: 21533121
9.  Time-resolved structural studies of protein reaction dynamics: a smorgasbord of X-ray approaches 
Time-resolved structural studies of proteins have undergone several significant developments during the last decade. Recent developments using time-resolved X-ray methods, such as time-resolved Laue diffraction, low-temperature intermediate trapping, time-resolved wide-angle X-ray scattering and time-resolved X-ray absorption spectroscopy, are reviewed.
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.
PMCID: PMC2824530  PMID: 20164644
time-resolved diffraction; structural biology; protein structural dynamics; Laue diffraction; kinetic crystallography; WAXS; XAS
10.  Structure of a photosynthetic reaction centre determined by serial femtosecond crystallography 
Nature Communications  2013;4:2911.
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.
Serial femtosecond crystallography is an X-ray free-electron-laser-based method that uses X-ray bursts to determine protein structures. Here the authors present the structure of a photosynthetic reaction centre, an integral membrane protein, achieved with no sign of X-ray-induced radiation damage.
PMCID: PMC3905732  PMID: 24352554

Results 1-10 (10)