Protein Structure Prediction

Práce popisuje prostorovou strukturu molekul bílkovin a databází uchovávajících representace této struktury, či její hierarchické klasifikace. Je poskytnut přehled současných metod výpočetní predikce struktury bílkovin, přičemž největší pozornost je soustředěna na komparativní modelování. Tato metoda je rovněž v základní podobě implementována a na závěr její implementace analyzována.This work describes the three dimensional structure of protein molecules and biological databases used to store information about this structure or its hierarchical classification. Current methods of computational structure prediction are overviewed with an emphasis on comparative modeling. This particular method is also implemented in a proof-of-concept program and finally, the implementation is analysed.

Sub-atomic resolution X-ray crystallography and neutron crystallography: promise, challenges and potential

The International Year of Crystallography saw the number of macromolecular structures deposited in the Protein Data Bank cross the 100000 mark, with more than 90000 of these provided by X-ray crystallography. The number of X-ray structures determined to sub-atomic resolution (i.e. ≤1 Å) has passed 600 and this is likely to continue to grow rapidly with diffraction-limited synchrotron radiation sources such as MAX-IV (Sweden) and Sirius (Brazil) under construction. A dozen X-ray structures have been deposited to ultra-high resolution (i.e. ≤0.7 Å), for which precise electron density can be exploited to obtain charge density and provide information on the bonding character of catalytic or electron transfer sites. Although the development of neutron macromolecular crystallography over the years has been far less pronounced, and its application much less widespread, the availability of new and improved instrumentation, combined with dedicated deuteration facilities, are beginning to transform the field. Of the 83 macromolecular structures deposited with neutron diffraction data, more than half (49/83, 59%) were released since 2010. Sub-mm3 crystals are now regularly being used for data collection, structures have been determined to atomic resolution for a few small proteins, and much larger unit-cell systems (cell edges >100 Å) are being successfully studied. While some details relating to H-atom positions are tractable with X-ray crystallography at sub-atomic resolution, the mobility of certain H atoms precludes them from being located. In addition, highly polarized H atoms and protons (H+) remain invisible with X-rays. Moreover, the majority of X-ray structures are determined from cryo-cooled crystals at 100 K, and, although radiation damage can be strongly controlled, especially since the advent of shutterless fast detectors, and by using limited doses and crystal translation at micro-focus beams, radiation damage can still take place. Neutron crystallography therefore remains the only approach where diffraction data can be collected at room temperature without radiation damage issues and the only approach to locate mobile or highly polarized H atoms and protons. Here a review of the current status of sub-atomic X-ray and neutron macromolecular crystallography is given and future prospects for combined approaches are outlined. New results from two metalloproteins, copper nitrite reductase and cytochrome c′, are also included, which illustrate the type of information that can be obtained from sub-atomic-resolution (∼0.8 Å) X-ray structures, while also highlighting the need for complementary neutron studies that can provide details of H atoms not provided by X-ray crystallography

Trends and Challenges in Experimental Macromolecular Crystallography

Macromolecular X-ray crystallography underpins the vigorous field of structural molecular biology having yielded many protein, nucleic acid and virus structures in fine detail. The understanding of the recognition by these macromolecules, as receptors, of their cognate ligands involves the detailed study of the structural chemistry of their molecular interactions. Also these structural details underpin the rational design of novel inhibitors in modern drug discovery in the pharmaceutical industry. Moreover, from such structures the functional details can be inferred, such as the biological chemistry of enzyme reactivity. There is then a vast number and range of types of biological macromolecules that potentially could be studied. The completion of the protein primary sequencing of the yeast genome, and the human genome sequencing project comprising some 105 proteins that is underway, raises expectations for equivalent three dimensional structural database

Determining molecular conformation from distance or density data

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2000.Includes bibliographical references (p. 126-130).This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.The determination of molecular structures is of growing importance in modern chemistry and biology. This thesis presents two practical, systematic algorithms for two structure determination problems. Both algorithms are branch-and-bound techniques adapted to their respective domains. The first problem is the determination of structures of multimers given rigid monomer structures and (potentially ambiguous) intermolecular distance measurements. In other words, we need to find the the transformations to produce the packing interfaces. A substantial difficulty results from ambiguities in assigning intermolecular distance measurements (from NMR, for example) to particular intermolecular interfaces in the structure. We present a rapid and efficient method to simultaneously solve the packing and the assignment problems. The algorithm, AmbiPack, uses a hierarchical division of the search space and the branch-and-bound algorithm to eliminate infeasible regions of the space and focus on the remaining space. The algorithm presented is guaranteed to find all solutions to a pre-determined resolution. The second problem is building a protein model from the initial three dimensional electron density distribution (density map) from X-ray crystallography. This problem is computationally challenging because proteins are extremely flexible.(cont.) Our algorithm, ConfMatch, solves this "map interpretation" problem by matching a detailed conformation of the molecule to the density map (conformational matching). This "best match" structure is defined as one which maximizes the sum of the density at atom positions. The most important idea of ConfMatch is an efficient method for computing accurate bounds for branch-and-bound search. Confmatch relaxes the conformational matching problem, a problem which can only be solved in exponential time (NP-hard), into one which can be solved in polynomial time. The solution to the relaxed problem is a guaranteed upper bound for the conformational matching problem. In most empirical cases, these bounds are accurate enough to prune the search space dramatically, enabling ConfMatch to solve structures with more than 100 free dihedral angles.by Cheuk-san (Edward) Wang.Ph.D

Data collection with a tailored X-ray beam size at 2.69 angstrom wavelength (4.6 keV):sulfur SAD phasing of Cdc23(Nterm)

The capability to reach wavelengths of up to 3.1 Å at the newly established EMBL P13 beamline at PETRA III, the new third-generation synchrotron at DESY in Hamburg, provides the opportunity to explore very long wavelengths to harness the sulfur anomalous signal for phase determination. Data collection at $\lambda$ = 2.69 Å (4.6 keV) allowed the crystal structure determination by sulfur SAD phasing of Cdc23$^{Nterm}$, a subunit of the multimeric anaphase-promoting complex (APC/C). At this energy, Cdc23$^{Nterm}$ has an expected Bijvoet ratio <|F$_{anom}$|>/<F> of 2.2%, with 282 residues, including six cysteines and five methionine residues, and two molecules in the asymmetric unit (65.4 kDa; 12 Cys and ten Met residues). Selectively illuminating two separate portions of the same crystal with an X-ray beam of 50 µm in diameter allowed crystal twinning to be overcome. The crystals diffracted to 3.1 Å resolution, with unit-cell parameters a = b = 61.2, c = 151.5 Å, and belonged to space group $P4_3$. The refined structure to 3.1 Å resolution has an R factor of 18.7% and an R$_{free}$ of 25.9%. This paper reports the structure solution, related methods and a discussion of the instrumentation

Surface and solvent influences on protein crystallization

The role of water in protein crystallization was explored by investigating the effects of three factors (salts, point mutations and pressure) on subtilisin crystallization.;Solubility and growth kinetics of three subtilisin mutants in three salt solutions were measured. The decrease of the solubility of Properase RTM and PurafectRTM subtilisin followed the reverse order of the Hofmeister series: SCN- \u3e NO3- \u3e Cl-. The solubility of ProperaseRTM was higher than other two mutants. Crystal morphology changed with the nature of salts and the substitution of surface residues. The required supersaturation (c-s)/s for a given growth rate increased when solubility was decreased. The effect of anion on protein growth was related to the molar Gibbs free energy of hydration of the anion.;Structural and energetic considerations for crystallization of two subtilisin mutants (ProperaseRTM and PurafectRTM) were compared. The average hydrophobicity, solvent accessible surface area (ASA) and the number of hydrogen bonds and salt bridges were calculated to quantify surface properties of proteins in intermolecular contact patches. All three amino acid substitutions are present in the contact patches. Properase RTM lattice involves more atomic contacts and hydrogen bonds and larger accessible surface area, which corresponding to the faster growth of ProperaseRTM crystals. Non-electrostatic interaction energy was calculated for each contact direction and the competition of misoriented molecules with correctly oriented ones was considered to explain the variation of growth kinetics;The increase of solubility with pressure gave a total volume change for crystallization of 37 cm3/mol, whereas the decrease of nucleation rate with pressure gave an activation volume for nucleation of 226 cm 3/mol. 983 water molecules were estimated to attend Properase RTM crystallization.;The second virial coefficients (B2) of Properase RTM and PurafectRTM subtilisin under crystallization conditions were measured by static light scattering as a function of salt type and salt concentration, showing that conditions with slight negative B2 are suitable for protein crystallization. A DLVO-type model was used to fit the effective Hamaker constants for subtilisin and solubility was quantitatively correlated with B2 using a theoretically based correlation

Systematic Correlation of Structural, Thermodynamic and Residual Solvation Properties of Hydrophobic Substituents in Hydrophobic Pockets Using Thermolysin as a Case Study

Water molecules participate besides protein and ligand as an additional binding partner in every in vivo protein–ligand binding process. The displacement of water molecules from apolar surfaces of solutes is considered the driving force of the hydrophobic effect. It is generally assumed that the mobility of the water molecules increases through the displacement, and, as a consequence, entropy increases. This explanation, which is based on experiments with simple model systems, is, however, insufficient to describe the hydrophobic effect as part of the highly complex protein–ligand complex formation process. For instance, the displacement of water molecules from apolar surfaces that already exhibit an increased mobility before their displacement can result in an enthalpic advantage. Furthermore, it has to be considered that by the formation of the protein–ligand complex a new solvent-exposed surface is created, around which water molecules have to rearrange. The present thesis focuses on the impact of the latter effect on the thermodynamic and kinetic binding properties of a given ligand. A congeneric ligand series comprised of nine ligands binding to the model protein thermolysin (TLN) was analyzed to determine the impact of the rearrangement of water molecules around the surface of a newly formed protein–ligand complex on the thermodynamic binding properties of a ligand. The protein–ligand complexes were characterized structurally by X-ray crystallography and thermodynamically by isothermal titration calorimetry (ITC). The only structural difference between the ligands was their strictly apolar P2’ substituent, which changed in size from a methyl to a phenylethyl group. The P2’ group interacts with the flat, apolar, and well-solvated S2’ pocket of TLN. Depending on the bound ligand, the solvent-exposed surface of the protein–ligand complex changes. The ITC measurements revealed strong thermodynamic differences between the different ligands. The structural analysis showed ligand-coating water networks pronounced to varying degrees. A pronounced water network clearly correlated with a favorable enthalpic and less favorable entropic term, and overall resulted in an affinity gain. Based on these results, new P2’ substituents were rationally designed with the aim to achieve stronger stabilization of the adjacent water networks and thereby further increase ligand affinity. First, the quality of the putative water networks was validated using molecular dynamics (MD) simulations. Subsequently, the proposed ligands were synthesized, crystallized in complex with TLN, and analyzed thermodynamically. Additionally, a kinetic characterization using surface plasmon resonance (SPR) was performed. The crystallographically determined water networks adjacent to the P2’ substituents were in line with their predictions conducted by MD simulations. The ligands showed increasingly pronounced water networks as well as a slight enthalpy-drive affinity increase compared to the ligands from the initial study. The ligand with the highest affinity showed an almost perfect water network as well as a significantly reduced dissociation constant. To analyze the influence of the ligand-coating water networks on the kinetic binding properties of a ligand, seventeen congeneric TLN ligands exhibiting different P2’ groups were kinetically (by SPR) and crystallographically characterized. The different degree of the water network stabilization showed only a minor influence on the binding kinetic properties. By contrast, the strength of the interaction between the ligand and Asn112 proved crucial for the magnitude of the dissociation rate constant. A strong interaction resulted in a considerably prolonged residence time of the ligand by hindering TLN to undergo a conformational transition that is necessary for ligand release. In the last study, the reason for the exceptionally high affinity gain for addressing the deep, apolar S1’ pocket of TLN with apolar ligand portions was investigated. Therefore, a congeneric TLN ligand series substituted with differently large apolar P1’ substituents (ranging from a single hydrogen atom to an iso-butyl group) was analyzed. The exchange of the hydrogen atom at the P1’ position with a single methyl group already results in a 100-fold affinity increase of the ligand. To elucidate the molecular mechanism behind this considerable affinity gain, the solvation state of the S1’ pocket was carefully analyzed. The results strongly indicate that the S1’ pocket is completely free of the presence of any water molecules. Thus, the huge affinity gain was attributed to the absence of an energetically costly desolvation step. The data presented in this thesis show that to describe the thermodynamic signature of the hydrophobic effect it is necessary to explicitly consider the change of the thermodynamic properties of every involved water molecule. Solely considering the buried apolar surface area and assigning an entropic term to it is not sufficient. The increasing stabilization of the water network adjacent to the protein-bound ligand represents a promising approach — quite independent of specific properties of the target protein — to optimize the thermodynamic profile of a given ligand. This approach also allows fine-tuning of the kinetic binding parameters

High Resolution X-ray and Neutron Crystallographic Studies of \u3cem\u3eEscherichia coli\u3c/em\u3e Dihydrofolate Reductase

Dihydrofolate Reductases (DHFRs) have been identified in nearly every proteome and are essential for most biosynthetic pathways involving one-carbon transfer reactions due to their recycling of tetrahydrofolate (THF). They catalyze the NADPH-dependent reduction of dihydrofolate (DHF), producing THF. Inhibition of DHFR ultimately depletes cellular pools of THF; causing a reduced supply of thymine nucleotides for DNA synthesis, resulting in genomic instability and cell death. Therefore, DHFRs remain important drug targets in antimicrobial and chemotherapeutic treatments. Despite exhaustive investigation of E. coli chromosomal DHFR, controversy persists over the dynamics of regulatory loops (the Met20, the βF-βG, and the βG-βH) and the nature of the interaction between methotrexate (MTX), a tight-binding anti-cancer drug, and Asp 27, the only ionizable residue in the active site. Also of importance is the ionization state of Asp 27 in the apoenzyme and other complexes. Hydrogen atoms (H) likely play a critical role in DHFR ligand binding and catalysis, yet are difficult to directly visualize. High resolution X-ray and neutron crystallography have been utilized in this dissertation to provide accurate positions of H within the DHFR active site and to probe dynamics of the enzyme. The ultrahigh resolution X-ray structures of DHFR/MTX (1.0Å; chapter 4), apo DHFR (1.05Å), and DHFR/MTX/NADPH (1.4Å; both chapter 5) have been solved. Novel features were observed in the electron density maps, including the ability to model the Met20 loop in the apoenzyme as closed (reported disordered previously) and alternate side chain conformations in all the structures. The high data-to-parameter ratio of the apoenzyme and the MTX data sets allowed anisotropic B-factor refinement and full-matrix refinement to calculate carboxylate bond lengths and estimates of their deviations. The apoenzyme has highly different bond lengths for its Asp 27 carboxylate, thus, it is neutral at physiological pH. The carboxylate bond lengths of the Asp 27 in both the monomers of the asymmetric unit of the DHFR/MTX crystal are nearly equal, suggesting it is charged at physiological pH. If H is substituted for deuterium (D), neutrons are especially powerful probes due to D’s strong positive scattering length. To assign protonation states to the MTX and the Asp 27 by the direct identification of D, a neutron structure has been solved to 2.2Å resolution from nearly 80% complete data collected on a 0.3mm3 crystal (chapter 4). Prerequisite to the neutron experiment was the growth and D2O-soaking of large-volume crystals (chapter 3). The DHFR/MTX cocrystal possesses the largest primitive unit cell and is the smallest D2O-soaked crystal used successfully in a neutron diffraction experiment. This is the 11th novel protein ever to be solved by neutron crystallography (the 16th total structure). Nearly 2/3 of the amide backbone has undergone H/D exchange, an indicator of protein dynamics. However, monomer B, where the Met20 loop is closed, is ~10% more exchanged than monomer A, where the Met20 loop is partially occluded. Based on results from D occupancy refinement and analysis of the neutron maps, it is concluded that the MTX N1 is protonated when bound to DHFR. Paired with the X-ray data, this is new strong evidence that the Asp 27·MTX interaction is ionic in nature. To increase the signal-to-noise ratio in future neutron experiments, perdeuterated protein has been produced and its D enrichment measured by mass spectrometry. X-ray data (to 1.2Å) has now been collected on a perdeuterated DHFR/MTX cocrystal and it is isomorphous to the native cocrystals (chapter 3)

Study of ligand-based virtual screening tools in computer-aided drug design

Virtual screening is a central technique in drug discovery today. Millions of molecules can be tested in silico with the aim to only select the most promising and test them experimentally. The topic of this thesis is ligand-based virtual screening tools which take existing active molecules as starting point for finding new drug candidates. One goal of this thesis was to build a model that gives the probability that two molecules are biologically similar as function of one or more chemical similarity scores. Another important goal was to evaluate how well different ligand-based virtual screening tools are able to distinguish active molecules from inactives. One more criterion set for the virtual screening tools was their applicability in scaffold-hopping, i.e. finding new active chemotypes. In the first part of the work, a link was defined between the abstract chemical similarity score given by a screening tool and the probability that the two molecules are biologically similar. These results help to decide objectively which virtual screening hits to test experimentally. The work also resulted in a new type of data fusion method when using two or more tools. In the second part, five ligand-based virtual screening tools were evaluated and their performance was found to be generally poor. Three reasons for this were proposed: false negatives in the benchmark sets, active molecules that do not share the binding mode, and activity cliffs. In the third part of the study, a novel visualization and quantification method is presented for evaluation of the scaffold-hopping ability of virtual screening tools.Siirretty Doriast