Neutron Laue macromolecular crystallography

Recent progress in neutron protein crystallography such as the use of the Laue technique and improved neutron optics and detector technologies have dramatically improved the speed and precision with which neutron protein structures can now be determined. These studies are providing unique and complementary insights on hydrogen and hydration in protein crystal structures that are not available from X-ray structures alone. Parallel improvements in modern molecular biology now allow fully (per)deuterated protein samples to be produced for neutron scattering that essentially eradicate the large-and ultimately limiting-hydrogen incoherent scattering background that has hampered such studies in the past. High quality neutron data can now be collected to near atomic resolution (approximately 2.0 A) for proteins of up to approximately 50 kDa molecular weight using crystals of volume approximately 0.1 mm3 on the Laue diffractometer at ILL. The ability to flash-cool and collect high resolution neutron data from protein crystals at cryogenic temperature (15 K) has opened the way for kinetic crystallography on freeze trapped systems. Current instrument developments now promise to reduce crystal volume requirements by a further order of magnitude, making neutron protein crystallography a more accessible and routine technique.     

X-Ray-Radiation-Induced Cooperative Atomic Movements in Protein

X-rays interact with biological matter and cause damage. Proteins and other macromolecules are damaged primarily by ionizing X-ray photons and secondarily by reactive radiolytic chemical species. In particular, protein molecules are damaged during X-ray diffraction experiments with protein crystals, which is, in many cases, a serious hindrance to structure solution. The local X-ray-induced structural changes of the protein molecule have been studied using a number of model systems. However, it is still not well understood whether these local chemical changes lead to global structural changes in protein and what the mechanism is.We present experimental evidence at atomic resolution indicating the movement of large parts of the protein globule together with bound water molecules in the early stages of radiation damage to the protein crystal. The data were obtained from a crystal cryocooled to ~ 100 K and diffracting to 1 ?. The movement of the protein structural elements occurs simultaneously with the decarboxylation of several glutamate and aspartate residues that mediate contacts between moving protein structural elements and with the rearrangement of the water network. The analysis of the anisotropy of atomic displacement parameters reveals that the observed atomic movements occur at different rates in different unit cells of the crystal. Thus, the examination of the cooperative atomic movement enables us to better understand how radiation-induced local chemical and structural changes of the protein molecule eventually lead to disorder in protein crystals.     

Crystallization and preliminary X-ray diffraction analysis of staphylococcal enterotoxin type C.

The Type C staphylococcal enterotoxin produced by Staphylococcus aureus strain FRI-909 has been crystallized using a combination of two precipitants, ammonium sulfate and polyethylene glycol 400, with the addition of small amounts of detergent. Two related crystal forms have been obtained, one triclinic, and one tetragonal, both with one toxin molecule per asymmetric unit. These crystals are stable for at least 75 hr in the X-ray beam and diffract to at least 2.2 and 2.6 A, respectively. The triclinic crystals have unit cell parameters a = 38.5 A, b = 43.7 A, c = 36.9 A, and interaxial angles alpha = 99.9 degrees, beta = 95.8 degrees, and gamma = 98.5 degrees. The tetragonal crystals are of space group P4(1)22 with unit cell parameters a = 43.4 A and c = 278.0 A.     

Temperature dependence of protein dynamics: computer simulation analysis of neutron scattering properties

The temperature dependence of the internal dynamics of an isolated protein, bovine pancreatic trypsin inhibitor, is examined using normal mode analysis and molecular dynamics (MD) simulation. It is found that the protein exhibits marked anharmonic dynamics at temperatures of approximately 100-120 K, as evidenced by departure of the MD-derived average mean square displacement from that of the harmonic model. This activation of anharmonic dynamics is at lower temperatures than previously detected in proteins and is found in the absence of solvent molecules. The simulation data are also used to investigate neutron scattering properties. The effects are determined of instrumental energy resolution and of approximations commonly used to extract mean square displacement data from elastic scattering experiments. Both the presence of a distribution of mean square displacements in the protein and the use of the Gaussian approximation to the dynamic structure factor lead to quantified underestimation of the mean square displacement obtained.     

Fluctuations and correlations in crystalline protein dynamics: a simulation analysis of staphylococcal nuclease

Understanding collective motions in protein crystals is likely to furnish insight into functional protein dynamics and will improve models for refinement against diffraction data. Here, four 10 ns molecular dynamics simulations of crystalline Staphylococcal nuclease are reported and analyzed in terms of fluctuations and correlations in atomic motion. The simulation-derived fluctuations strongly correlate with, but are slightly higher than, the values derived from the experimental B-factors. Approximately 70% of the atomic fluctuations are due to internal protein motion. For 65% of the protein atoms the internal fluctuations converge on the nanosecond timescale. Convergence is much slower for the elements of the interatomic displacement correlation matrix--of these, >80% converge within 1 ns for interatomic distances less, approximately <6 A, but only 10% for separations approximately =12 A. Those collective motions that converged on the nanosecond timescale involve mostly correlations within the beta-barrel or between alpha-helices of the protein. The R-factor with the experimental x-ray diffuse scattering for the crystal, which is determined by the displacement variance-covariance matrix, decreases to 8% after 10 ns simulation. Both the number of converged correlation matrix elements and the R-factor depend logarithmically on time, consistent with a model in which the number of energy minima sampled depends exponentially on the maximum energy barrier crossed. The logarithmic dependence is also extrapolated to predict a convergence time for the whole variance-covariance matrix of approximately 1 micros.     

Modeling protein conformational changes by iterative fitting of distance constraints using reoriented normal modes

Recently we have developed a normal-modes-based algorithm that predicts the direction of protein conformational changes given the initial state crystal structure together with a small number of pairwise distance constraints for the end state. Here we significantly extend this method to accurately model both the direction and amplitude of protein conformational changes. The new protocol implements a multisteps search in the conformational space that is driven by iteratively minimizing the error of fitting the given distance constraints and simultaneously enforcing the restraint of low elastic energy. At each step, an incremental structural displacement is computed as a linear combination of the lowest 10 normal modes derived from an elastic network model, whose eigenvectors are reorientated to correct for the distortions caused by the structural displacements in the previous steps. We test this method on a list of 16 pairs of protein structures for which relatively large conformational changes are observed (root mean square deviation >3 angstroms), using up to 10 pairwise distance constraints selected by a fluctuation analysis of the initial state structures. This method has achieved a near-optimal performance in almost all cases, and in many cases the final structural models lie within root mean square deviation of 1 approximately 2 angstroms from the native end state structures.     

Crystal structure of human serum albumin at 2.5 A resolution.

A new triclinic crystal form of human serum albumin (HSA), derived either from pool plasma (pHSA) or from a Pichia pastoris expression system (rHSA), was obtained from polyethylene glycol 4000 solution. Three-dimensional structures of pHSA and rHSA were determined at 2.5 A resolution from the new triclinic crystal form by molecular replacement, using atomic coordinates derived from a multiple isomorphous replacement work with a known tetragonal crystal form. The structures of pHSA and rHSA are virtually identical, with an r.m. s. deviation of 0.24 A for all Calpha atoms. The two HSA molecules involved in the asymmetric unit are related by a strict local twofold symmetry such that the Calpha atoms of the two molecules can be superimposed with an r.m.s. deviation of 0.28 A in pHSA. Cys34 is the only cysteine with a free sulfhydryl group which does not participate in a disulfide linkage with any external ligand. Domains II and III both have a pocket formed mostly of hydrophobic and positively charged residues and in which a very wide range of compounds may be accommodated. Three tentative binding sites for long-chain fatty acids, each with different surroundings, are located at the surface of each domain.     

Crystallization and preliminary X-ray analysis of a peridinin-chlorophyll a protein from Amphidinium carterae

Crystals of a water-soluble (Mr approximately 39,000) peridinin-chlorophyll a protein from Amphidinium carterae are reported. The crystals diffract to 2.2 A and belong to a monoclinic (B2) and a triclinic (P1) space group. Spectra of the protein in the crystal and in solution are almost identical.     

Structures of triclinic mono- and di-N-acetylglucosamine: lysozyme complexes--a crystallographic study.

N-acetylglucosamine and di-N-acetylglucosamine bind to lysozyme in the triclinic crystal form. Attempts to bind tri-N-acetylglucosamine were unsuccessful. Difference syntheses showed both GalNAc³ and di-GalNAc to be bound as the β-anomers, and revealed shifts in the positions of some of the lysozyme atoms. As was found in the binding studies with tetragonal lysozyme, the side chain of Trp62 moved toward the bound inhibitor. The carbonyl oxygen atom of Ala107 appeared to shift slightly, but there was no suggestion of movement in the lobes of the molecules as was evident in the tetragonal crystals.     

Toward chaperone-assisted crystallography: protein engineering enhancement of crystal packing and X-ray phasing capabilities of a camelid single-domain antibody (VHH) scaffold

A crystallization chaperone is an auxiliary protein that binds to a target of interest, enhances and modulates crystal packing, and provides high-quality phasing information. We critically evaluated the effectiveness of a camelid single-domain antibody (V(H)H) as a crystallization chaperone. By using a yeast surface display system for V(H)H, we successfully introduced additional Met residues in the core of the V(H)H scaffold. We identified a set of SeMet-labeled V(H)H variants that collectively produced six new crystal forms as the complex with the model antigen, RNase A. The crystals exhibited monoclinic, orthorhombic, triclinic, and tetragonal symmetry and have one or two complexes in the asymmetric unit, some of which diffracted to an atomic resolution. The phasing power of the Met-enriched V(H)H chaperone allowed for auto-building the entire complex using single-anomalous dispersion technique (SAD) without the need for introducing SeMet into the target protein. We show that phases produced by combining SAD and V(H)H model-based phases are accurate enough to easily solve structures of the size reported here, eliminating the need to collect multiple wavelength multiple-anomalous dispersion (MAD) data. Together with the presence of high-throughput selection systems (e.g., phage display libraries) for V(H)H, the enhanced V(H)H domain described here will be an excellent scaffold for producing effective crystallization chaperones.     

Effect of the environment on the protein dynamical transition: a neutron scattering study

We performed an elastic neutron scattering investigation of the molecular dynamics of lysozyme solvated in glycerol, at different water contents h (grams of water/grams of lysozyme). The marked non-Gaussian behavior of the elastic intensity was studied in a wide experimental momentum transfer range, as a function of the temperature. The internal dynamics is well described in terms of the double-well jump model. At low temperature, the protein total mean square displacements exhibit an almost linear harmonic trend irrespective of the hydration level, whereas at the temperature T(d) a clear changeover toward an anharmonic regime marks a protein dynamical transition. The decrease of T(d) from approximately 238 K to approximately 195 K as a function of h is reminiscent of that found in the glass transition temperature of aqueous solutions of glycerol, thus suggesting that the protein internal dynamics as a whole is slave to the environment properties. Both T(d) and the total mean square displacements indicate that the protein flexibility strongly rises between 0.1 and 0.2h. This hydration-dependent dynamical activation, which is similar to that of hydrated lysozyme powders, is related to the specific interplay of the protein with the surrounding water and glycerol molecules.     

Protein-water displacement distributions

The statistical properties of fast protein-water motions are analyzed by dynamic neutron scattering experiments. Using isotopic exchange, one probes either protein or water hydrogen displacements. A moment analysis of the scattering function in the time domain yields model-independent information such as time-resolved mean square displacements and the Gauss-deviation. From the moments, one can reconstruct the displacement distribution. Hydration water displays two dynamical components, related to librational motions and anomalous diffusion along the protein surface. Rotational transitions of side chains, in particular of methyl groups, persist in the dehydrated and in the solvent-vitrified protein structure. The interaction with water induces further continuous protein motions on a small scale. Water acts as a plasticizer of displacements, which couple to functional processes such as open-closed transitions and ligand exchange.     

Flexibility in crystalline insulins.

Comparisons of atomic models for chemically identical protein molecules solved in differing crystal environments provide information on flexibility in the protein structure. The structures of five T4 lysozyme proteins in differing crystal environments showed large relative displacements of the two domains with conserved backbone conformations that are connected by a flexible hinge (H. R. Faber and B. W. Matthews. 1990. Nature (Lond.). 348:263-266). In contrast, my comparison of the positions of all the atoms in two crystal forms of insulin shows that the structural changes caused by the differing crystal contacts are contained within nearby amino acids and are not propagated through the core of the insulin molecule. Groups of atoms that are most significantly displaced are not shifted in large rigid units but are repacked into new and distinct conformations. The transmission of displacements through the single domain insulin molecule is, like the movements due to thermal vibrations (D. L. D. Caspar, J. Clarage, D. M. Salunke, M. S. Clarage. 1988. Nature (Lond.). 332:659-662), characterized by short-range interactions between small atomic groups.     

Analysis of phi and chi 1 torsion angles for hen lysozyme in solution from 1H NMR spin-spin coupling constants

Three-bond 3JHN alpha coupling constants have been determined for 106 residues and 3J alpha beta coupling constants have been measured for 57 residues of the 129-residue protein hen egg white lysozyme. These NMR data have been compared with torsion angles defined in the tetragonal and the triclinic crystal forms of the protein. For most residues the measured 3JHN alpha values were consistent with the phi torsion angles found in both crystal forms; the RMS difference between the coupling constants calculated by using the tetragonal crystal structure phi angles and the experimental 3JHN alpha values is 0.88 Hz. Thus there appears to be no significant averaging of the phi torsion angle either in the interior or at the surface of the protein. For 41 of the residues where 3J alpha beta coupling constants have been determined, the values are consistent with a single staggered conformation about the chi 1 torsion angle and there is complete agreement between the NMR data in solution and the torsion angles defined in the crystalline state. In contrast, for the other 16 residues where 3J alpha beta coupling constant values have been measured, the data indicate extensive motional averaging about the chi 1 torsion angle. These residues occur largely on the surface of the protein and examination of the crystal structures shows that many of these residues adopt a different conformation in the triclinic and tetragonal crystal forms and have high crystallographic temperature factors. It appears, however, that in solution conformational flexibility of the side chains of surface residues is significantly more pronounced than in individual crystal structures.     

Crystal versus solution structures of thiamine diphosphate-dependent enzymes

The quaternary structures of the thiamine diphosphate-dependent enzymes transketolase (EC 2.2.1.1; from Saccharomyces cerevisiae), pyruvate oxidase (EC 1.2.3.3; from Lactobacillus plantarum), and pyruvate decarboxylase (EC 4.1.1.1; from Zymomonas mobilis and brewers' yeast, the latter in the native and pyruvamide-activated forms) were examined by synchrotron x-ray solution scattering. The experimental scattering data were compared with the curves calculated from the crystallographic models of these multisubunit enzymes. For all enzymes noted above, except the very compact pyruvate decarboxylase from Z. mobilis, there were significant differences between the experimental and calculated profiles. The changes in relative positions of the subunits in solution were determined by rigid body refinement. For pyruvate oxidase and transketolase, which have tight intersubunit contacts in the crystal, relatively small modifications of the quaternary structure (root mean square displacements of 0.23 and 0.27 nm, respectively) sufficed to fit the experimental data. For the enzymes with looser contacts (the native and activated forms of yeast pyruvate decarboxylase), large modifications of the crystallographic models (root mean square displacements of 0.58 and 1.53 nm, respectively) were required. A clear correlation was observed between the magnitude of the distortions induced by the crystal environment and the interfacial area between subunits.     

A nuclear magnetic resonance study of the hydrogen-exchange behaviour of lysozyme in crystals and solution

Amide hydrogen/deuterium exchange behaviour has been studied for all of the peptide amides of hen lysozyme by means of two-dimensional n.m.r. spectroscopy. The amides have been grouped into four categories on the basis of their rates of exchange in solution at pH 4.2 and 7.5. The distribution of the amides into the different categories has been examined in the light of the crystallographic structural information, considering the type of secondary structure, the nature of hydrogen bonding and the distance from the protein surface. None of these features was found to determine uniquely the pattern of hydrogen exchange rates within the protein. The exchange behaviour of the individual amides could, however, in general be rationalized by a combination of these features. Hydrogen exchange was also monitored in both tetragonal and triclinic crystals of lysozyme, by allowing exchange to take place in the crystals prior to dissolution and recording of n.m.r. spectra under conditions where further exchange was minimized. This enabled direct comparison to be made of the exchange behaviour in the crystals and solution. A reduction in exchange rate was observed in the crystalline state relative to solution for a substantial number of amides and distinct differences between exchange in the different crystals could be observed. These differences between the solution and the different crystal states do not, however, correlate in a simple manner with proximity to intermolecular contacts in the crystals. However, the existence of these contacts, which are on the surface of the protein molecule, have a profound effect on the exchange of amides in the interior of the protein. The results indicate that the spectrum of fluctuations giving rise to hydrogen exchange may be significantly altered by the intermolecular interactions present within the crystalline state.     

Protein dynamics from X-ray crystallography: anisotropic, global motion in diffuse scattering patterns

Understanding X-ray crystallographic diffuse scattering is likely to improve our comprehension of equilibrium collective protein dynamics. Here, using molecular dynamics (MD) simulation, a detailed analysis is performed of the origins of diffuse scattering in crystalline Staphylococcal nuclease, for which the complete diffuse scattering pattern has been determined experimentally. The hydrogen-atom contribution and the scattering range over which the scattering can be considered to be a sum of solvent and protein scattering are determined. Two models of correlated protein motion are investigated by calculating the model-derived diffuse scattering and comparing with the scattering calculated directly from MD trajectories. In one model, previously used in diffuse scattering interpretation, the atomic displacement correlations decay isotropically with increasing separation. Model correlation lengths are obtained by refining the model scattering against the simulation-derived scattering pattern, and are found to be significantly different from those correlation lengths derived directly from the MD trajectories. Furthermore, the convergence between the model-derived and MD-derived scattering is poor. The second model, in which the displacement correlations are calculated from the principal components of the MD trajectories, is capable of fully reproducing the MD-derived diffuse scattering if the approximately 50% lowest-frequency modes are included. However, a small number ( approximately 10) of lowest-frequency and largest-amplitude modes dominates the diffuse scattering and thus the correlated protein motions. A detailed analysis of the principal components is performed. In particular, the effective free energy profile associated with each principle mode is analyzed and the eigenfrequency and damping coefficient computed using a model of Brownian dynamics. Those collective modes with effective frequencies below approximately 0.5 THz, including those that determine the diffuse scattering, are overdamped.     

Crystallization and preliminary X-ray diffraction studies of the Lb proteinase from foot-and-mouth disease virus.

Different crystal forms of the C23A mutant from the leader proteinase of foot-and-mouth disease virus were obtained by the hanging drop vapor diffusion technique, using MgCl2 and PEG 6000 as precipitants. Well-developed crystals, with cubic morphology growing to approximately 1.0 mm3 in size, presented a large unit cell parameter of 274.5 A and diffracted to, at most, 5 A resolution. A second type of crystal had a tetragonal appearance and these were obtained in droplets soaked in a silica gel matrix. These crystals, with an approximate size of 0.3 X 0.3 X 0.7 mm3, diffracted to approximately 4.0 A resolution, but presented a strong anisotropic mosaicity around the longest crystal axis. Crystals with a needlelike morphology and reaching sizes of about 0.2 X 0.3 X 1.2 mm3 diffracted beyond 3.5 A resolution and were stable to X-ray radiation for approximately one day when using a conventional source at room temperature. These crystals are orthorhombic with space group I222 (or I2(1)2(1)2(1)) and unit cell dimensions a = 65.9 A, b = 104.3 A, and c = 124.0 A, and appear well suited for high-resolution studies. Density packing considerations are consistent with the presence of two molecules in the asymmetric unit and a solvent content of approximately 54%.     

Side-necked turtle (Pleurodira, Chelonia, reptilia) hemoglobin: cDNA-derived primary structures and X-ray crystal structures of Hb A

Red blood cells of yellow-spotted river turtles (Podocnemis unifilis, Pleurodira, Chelonia, REPTILIA) have two hemoglobin (Hb) components, Hb A and Hb D. We purified the hemoglobin component homologous to amniote (reptiles, birds, and mammals) adult Hb A which comprises two identical α(A) -globin polypeptides and two identical β-globin polypeptides. To establish the crystal structure of Podocnemis Hb A, we first determined the globin primary structures using cDNA nucleotide sequencing with the assistance of protein sequencing. The purified Podocnemis Hb A produced a different form of crystal for each of the two different buffer systems used: form A, tetragonal crystals (space group, P4?2?2), produced under neutral pH (pH 7-8) conditions; and form B, hexagonal crystals (space group, P6?22), produced under high alkaline pH (pH 11-13) conditions. Single crystals of the two forms were examined by Raman microscopy with an excitation of 532 nm, indicating their structural differences. The crystal structures of the two forms were constructed by X-ray crystallographic diffraction at a resolution of 2.20 ? for form A and 2.35 ? for form B. The differences of the tertiary and quaternary structures of the two forms were marginal; however, one clear difference was found in helix structure. When comparing Podocnemis Hb A with Hb A from specimens in other taxa, such as Anser indicus (birds) and Homo sapiens (mammals) by SHELXPRO, the root mean square deviation (RMSD) between the corresponding Cα atoms of the two globins does not exceed 2.0 ?. These low values indicate the crystal structures resemble each other. Our data on X-ray crystal structures and Raman spectra not only reveal the first findings on the two crystal forms of Podocnemis unifilis Hb A but also provide the first refined models for reptilian adult Hb A.     

Neutron scattering reveals the dynamic basis of protein adaptation to extreme temperature

To explore protein adaptation to extremely high temperatures, two parameters related to macromolecular dynamics, the mean square atomic fluctuation and structural resilience, expressed as a mean force constant, were measured by neutron scattering for hyperthermophilic malate dehydrogenase from Methanococcus jannaschii and a mesophilic homologue, lactate dehydrogenase from Oryctolagus cunniculus (rabbit) muscle. The root mean square fluctuations, defining flexibility, were found to be similar for both enzymes (1.5 A) at their optimal activity temperature. Resilience values, defining structural rigidity, are higher by an order of magnitude for the high temperature-adapted protein (0.15 Newtons/meter for O. cunniculus lactate dehydrogenase and 1.5 Newtons/meter for M. jannaschii malate dehydrogenase). Thermoadaptation appears to have been achieved by evolution through selection of appropriate structural rigidity in order to preserve specific protein structure while allowing the conformational flexibility required for activity.