Modeling the hydration of proteins: prediction of structural and hydrodynamic parameters from X-ray diffraction and scattering data

The implications of protein-water interactions are of importance for understanding the solution behavior of proteins and for analyzing the fine structure of proteins in aqueous solution. Starting from the atomic coordinates, by bead modeling the scattering and hydrodynamic properties of proteins can be predicted reliably (Debye modeling, program HYDRO). By advanced modeling techniques the hydration can be taken into account appropriately: by some kind of rescaling procedures, by modeling a water shell, by iterative comparisons to experimental scattering curves (ab initio modeling) or by special hydration algorithms. In the latter case, the surface topography of proteins is visualized in terms of dot surface points, and the normal vectors to these points are used to construct starting points for placing water molecules in definite positions on the protein envelope. Bead modeling may then be used for shaping the individual atomic or amino acid residues and also for individual water molecules. Among the tuning parameters, the choice of the scaling factor for amino acid hydration and of the molecular volume of bound water turned out to be crucial. The number and position of bound water molecules created by our hydration modeling program HYDCRYST were compared with those derived from X-ray crystallography, and the capability to predict hydration, structural and hydrodynamic parameters (hydrated volume, radius of gyration, translational diffusion and sedimentation coefficients) was compared with the findings generated by the water-shell approach CRYSOL. If the atomic coordinates are unknown, ab initio modeling approaches based on experimental scattering curves can provide model structures for hydrodynamic predictions.     

Secondary structure of condensed DNA. wide-angle, small-angle x-ray scattering and circular dichroism

Ethanol precipitated DNA shows a CD spectrum of the +psi-type which is similar to that of DNA in the A-form. DNA condensed with cetyl-trimethylammonium-bromide shows, depending on the condensation velocity, a CD spectrum of the -psi-type, or a CD spectrum only slightly modified from that of DNA in solution. The first spectrum is similar to that of DNA in the C-form, and the second one, to that of DNA in the B-form. Using large-angle X-ray scattering of the three DNA condensates and comparing them with the scattering curves calculated from the atom coordinates for the A-, B-, and C-form of DNA it is shown that the secondary structure of the DNA belongs in all three cases to the B-family. It follows from this result that the secondary structure of DNA alone does not determine the type of CD spectrum. The CD spectrum of condensed DNA is essentially determined by the supramolecular structures of the partially crystalline DNA condensates. These supramolecular structures can be demonstrated by the small-angle X-ray diagrams. The condensation of DNA by ethanol and cetyl-trimethylammonium-bromide proceeds in the form of a partial crystallization of the DNA.     

Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering

We demonstrate tracking of protein structural changes with time-resolved wide-angle X-ray scattering (TR-WAXS) with nanosecond time resolution. We investigated the tertiary and quaternary conformational changes of human hemoglobin under nearly physiological conditions triggered by laser-induced ligand photolysis. We also report data on optically induced tertiary relaxations of myoglobin and refolding of cytochrome c to illustrate the wide applicability of the technique. By providing insights into the structural dynamics of proteins functioning in their natural environment, TR-WAXS complements and extends results obtained with time-resolved optical spectroscopy and X-ray crystallography.     

Possibility of wide-angle neutron scattering in the study of proteins and nucleic acids in solutions

A method is proposed for calculating wide-angle neutron scattering curves of biopolymers at any fraction of heavy water (D2O) in solution. The method permits to accurately take into account the phenomenon of deuteroexchange. By this method neutron scattering curves of proteins and DNA have been calculated. The calculations have shown that at optimal fractions of D2O in solution the profiles of neutron scattering curves and their sensitivity to conformational rearrangements in protein molecules turned out to differ very little from those of corresponding X-ray curves. Thus the neutron scattering curves do not contain any additional information (as compared with those contained in X-ray scattering curves) on the structure of proteins in solution. On the contrary, neutron and X-ray scattering curves of DNA differ significantly at all fractions of D2O in solution and therefore the methods of wide-angle neutron and X-ray scattering could become mutually complementary in studying the structure of nucleic acids in solution.     

Lactobacillus surface layer proteins: structure, function and applications

Bacterial surface (S) layers are the outermost proteinaceous cell envelope structures found on members of nearly all taxonomic groups of bacteria and Archaea. They are composed of numerous identical subunits forming a symmetric, porous, lattice-like layer that completely covers the cell surface. The subunits are held together and attached to cell wall carbohydrates by non-covalent interactions, and they spontaneously reassemble in vitro by an entropy-driven process. Due to the low amino acid sequence similarity among S-layer proteins in general, verification of the presence of an S-layer on the bacterial cell surface usually requires electron microscopy. In lactobacilli, S-layer proteins have been detected on many but not all species. Lactobacillus S-layer proteins differ from those of other bacteria in their smaller size and high predicted pI. The positive charge in Lactobacillus S-layer proteins is concentrated in the more conserved cell wall binding domain, which can be either N- or C-terminal depending on the species. The more variable domain is responsible for the self-assembly of the monomers to a periodic structure. The biological functions of Lactobacillus S-layer proteins are poorly understood, but in some species S-layer proteins mediate bacterial adherence to host cells or extracellular matrix proteins or have protective or enzymatic functions. Lactobacillus S-layer proteins show potential for use as antigen carriers in live oral vaccine design because of their adhesive and immunomodulatory properties and the general non-pathogenicity of the species.     

Modeling large regions in proteins: applications to loops, termini, and folding

Template-based methods for predicting protein structure provide models for a significant portion of the protein but often contain insertions or chain ends (InsEnds) of indeterminate conformation. The local structure prediction "problem" entails modeling the InsEnds onto the rest of the protein. A well-known limit involves predicting loops of ≤12 residues in crystal structures. However, InsEnds may contain as many as ~50 amino acids, and the template-based model of the protein itself may be imperfect. To address these challenges, we present a free modeling method for predicting the local structure of loops and large InsEnds in both crystal structures and template-based models. The approach uses single amino acid torsional angle "pivot" moves of the protein backbone with a C(β) level representation. Nevertheless, our accuracy for loops is comparable to existing methods. We also apply a more stringent test, the blind structure prediction and refinement categories of the CASP9 tournament, where we improve the quality of several homology based models by modeling InsEnds as long as 45 amino acids, sizes generally inaccessible to existing loop prediction methods. Our approach ranks as one of the best in the CASP9 refinement category that involves improving template-based models so that they can function as molecular replacement models to solve the phase problem for crystallographic structure determination.     

Difference in the hydration water mobility around F-actin and myosin subfragment-1 studied by quasielastic neutron scattering

Hydration water is essential for a protein to perform its biological function properly. In this study, the dynamics of hydration water around F-actin and myosin subfragment-1 (S1), which are the partner proteins playing a major role in various cellular functions related to cell motility including muscle contraction, was characterized by incoherent quasielastic neutron scattering (QENS). The QENS measurements on the D₂O- and H₂O-solution samples of F-actin and S1 provided the spectra of hydration water, from which the translational diffusion coefficient (D_T), the residence time (τ_T), and the rotational correlation time (τ_R) were evaluated. The D_T value of the hydration water of S1 was found to be much smaller than that of the hydration water of F-actin while the τ_T values were similar between S1 and F-actin. On the other hand, the τ_R values of the hydration water of S1 was found to be larger than that of the hydration water of F-actin. It was also found that the D_T and τ_R values of the hydration water of F-actin are similar to those of bulk water. These results suggest a significant difference in mobility of the hydration water between S1 and F-actin: S1 has the typical hydration water, the mobility of which is reduced compared with that of bulk water, while F-actin has the unique hydration water, the mobility of which is close to that of bulk water rather than the typical hydration water around proteins.     

Accurate flexible fitting of high-resolution protein structures to small-angle x-ray scattering data using a coarse-grained model with implicit hydration shell

Small-angle x-ray scattering (SAXS) is a powerful technique widely used to explore conformational states and transitions of biomolecular assemblies in solution. For accurate model reconstruction from SAXS data, one promising approach is to flexibly fit a known high-resolution protein structure to low-resolution SAXS data by computer simulations. This is a highly challenging task due to low information content in SAXS data. To meet this challenge, we have developed what we believe to be a novel method based on a coarse-grained (one-bead-per-residue) protein representation and a modified form of the elastic network model that allows large-scale conformational changes while maintaining pseudobonds and secondary structures. Our method optimizes a pseudoenergy that combines the modified elastic-network model energy with a SAXS-fitting score and a collision energy that penalizes steric collisions. Our method uses what we consider a new implicit hydration shell model that accounts for the contribution of hydration shell to SAXS data accurately without explicitly adding waters to the system. We have rigorously validated our method using five test cases with simulated SAXS data and three test cases with experimental SAXS data. Our method has successfully generated high-quality structural models with root mean-squared deviation of 1 ∼ 3 Å from the target structures.     

Rotation isomerization of side chains of globular proteins and its effect on the x-ray scattering curves of proteins in a solution

A new method has been developed for averaging the intensity of X-ray diffuse scattering of proteins by different conformations of side groups. The method is based on the algorithm allowing to calculate statistical weights of rotation isomers of side chains. It is shown that for protein structures obtained from high resolution crystallographic data, conformations of the majority of surface groups correspond to rotation isomers with the greatest statistical weight. It has been found that for medium size proteins (with molecular weight varying from 15,000 to 30,000 dalton) whose structure has been determined at high resolution, the influence of rotation isomerization of side chains on the scattering indicatrices does not exceed 5%. The influence of side chains mobility on the scattering curves of large proteins is also small. For these two classes of proteins the rotation isomerization of side groups can be ignored when interpreting significant (exceeding 10%) divergences between experimental and theoretical scattering curves.     

Modeling the Hydration Layer around Proteins: HyPred

Protein hydration plays an integral role in determining protein function and stability. We develop a simple method with atomic level precision for predicting the solvent density near the surface of a protein. A set of proximal radial distribution functions are defined and calculated for a series of different atom types in proteins using all-atom, explicit solvent molecular dynamic simulations for three globular proteins. A major improvement in predicting the hydration layer is found when the protein is held immobile during the simulations. The distribution functions are used to develop a model for predicting the hydration layer with sub-1-Ångstrom resolution without the need for additional simulations. The model and the distribution functions for a given protein are tested in their ability to reproduce the hydration layer from the simulations for that protein, as well as those for other proteins and for simulations in which the protein atoms are mobile. Predictions for the density of water in the hydration shells are then compared with high occupancy sites observed in crystal structures. The accuracy of both tests demonstrates that the solvation model provides a basis for quantitatively understanding protein solvation and thereby predicting the hydration layer without additional simulations.     

Lateral order in gel, subgel and crystalline phases of lipid membranes: wide-angle X-ray scattering

The chain packing in ordered phases of lipid bilayers can be classified according to wide-angle X-ray diffraction (WAXS). For triclinic (T(‖)) and monoclinic (M(‖)) packing in crystalline L(c) phases, the wide-angle reflections index on an oblique lattice with spacings s(10)≠s(01)≠s(11), and each chain has four nearest neighbours. For orthorhombic (O(⊥)) packing in L(c) phases, and the rotationally symmetric equivalent in metastable low-temperature L(β) phases (mL(β)), the wide-angle reflections index on a centred rectangular lattice with spacings s(20)s(11), and each chain has two nearest neighbours. For hexagonal packing in the L(β), P(β') and interdigitated L(β)(i) gel phases, the wide-angle reflections index on a centred rectangular lattice with spacings s(20)=s(11), and each chain has six nearest neighbours. The available WAXS database for phospholipid and glycolipid bilayers is classified here according to the above scheme, by using well-established examples to assign the wide-angle reflections. The nearest and next-nearest neighbour chain-chain spacings, a(ch) and b(ch), and the cross-sectional area per chain, A(ch), are calculated for each phase of each lipid. These parameters determine many of the properties of the ordered lipid phases and, together with the chain tilt, specify the area occupied by the lipid head groups at the surface of the bilayer.     

Puromycin photoaffinity labels small- and large-subunit proteins at the A site of the Drosophila ribosome

[3H]Puromycin covalently incorporates into the protein and to a much lesser extent into the RNA components of Drosophila ribosomes in the presence of 254-nm light. The photoincorporation reaction takes place with a small number of large- (L2 and L17) and small- (S8 and S22) subunit proteins as determined by two-dimensional gel analysis. More quantitative one-dimensional gel results show that puromycin reacts with each of these proteins in a functional site specific manner. The small percentage of the total labeling that occurs with rRNA also appears to be site specific. The rRNA labeling arises from a puromycin-mediated cross-linking of ribosomal protein and rRNA. Ionic conditions shift the pattern of puromycin-labeled ribosomal proteins. These results suggest that puromycin can occupy two distinct sites on Drosophila 80S ribosomes. The pattern of ribosomal proteins labeled by puromycin is affected by the presence of other antibiotics such as emetine, anisomycin, and trichodermin.     

Addition of missing loops and domains to protein models by x-ray solution scattering

Inherent flexibility and conformational heterogeneity in proteins can often result in the absence of loops and even entire domains in structures determined by x-ray crystallographic or NMR methods. X-ray solution scattering offers the possibility of obtaining complementary information regarding the structures of these disordered protein regions. Methods are presented for adding missing loops or domains by fixing a known structure and building the unknown regions to fit the experimental scattering data obtained from the entire particle. Simulated annealing was used to minimize a scoring function containing the discrepancy between the experimental and calculated patterns and the relevant penalty terms. In low-resolution models where interface location between known and unknown parts is not available, a gas of dummy residues represents the missing domain. In high-resolution models where the interface is known, loops or domains are represented as interconnected chains (or ensembles of residues with spring forces between the C(alpha) atoms), attached to known position(s) in the available structure. Native-like folds of missing fragments can be obtained by imposing residue-specific constraints. After validation in simulated examples, the methods have been applied to add missing loops or domains to several proteins where partial structures were available.     

A Brillouin scattering study of the hydration of Li- and Na-DNA films

We have used Brillouin spectroscopy to study the velocities and attenuation of acoustic phonons in wet-spun films of Na-DNA and Li-DNA as a function of the degree of hydration at room temperature. Our data for the longitudinal acoustic (LA) phonon velocity vs water content display several interesting features and reveal effects that we can model at the atomic level as interhelical bond softening and relaxation of the hydration shell. The model for interhelical softening makes use of other physical parameters of these films, which we have determined by gravimetric, x-ray, and optical microscopy studies. We extract intrinsic elastic constants for hydrated Na-DNA molecules of c₁₁ ? 8.0 × 10¹⁰ dynes/cm² and c₃₃ ? 5.7 × 10¹⁰ dynes/cm², which corresponds to a Young's modulus, E ? 1.1 × 10¹⁰ dynes/cm² (with Poisson's ratio, σ = 0.44). The negative velocity anisotropy of the LA phonons indicates that neighboring DNA molecules are held together by strong interhelical bonds in the solid state. The LA phonon attenuation data can be understood by the relaxational model in which the acoustic phonon is coupled to a relaxation mode of the water molecules. Na-DNA undergoes the A to B phase transition at a relative humidity (rh) of 92% while Li-DNA (which remains in the B form in this range) decrystallizes at an rh of 84%. We find that our Brillouin results for Na- and Li-DNA are remarkably similar, indicating that the A to B phase transition does not play an important role in determining the acoustic properties of these two types of DNA.     

Techniques and applications of NMR to membrane proteins

The fact that membrane proteins are notoriously difficult to analyse using standard protocols for atomic-resolution structure determination methods have motivated adaptation of these techniques to membrane protein studies as well as development of new technologies. With this motivation, liquid-state nuclear magnetic resonance (NMR) has recently been used with success for studies of peptides and membrane proteins in detergent micelles, and solid-state NMR has undergone a tremendous evolution towards characterization of membrane proteins in native membrane and oriented phospholipid bilayers. In this mini-review, we describe some of the technological challenges behind these efforts and provide examples on their use in membrane biology.     

Study of the effect of hydration on the dynamics of various globular proteins by Rayleigh scattering of M?ssbauer radiation

Hydration relationships of the elastic scattering fraction of M?ssbauer radiation were studied for human serum albumin (HSA), pancreatic trypsin inhibitor and lysozyme within hydration degrees 0 less than or equal to h less than or equal to 0.75 g/g (at T = 295 degrees K) and temperatures 100K less than or equal to T less than or equal to 320 K (for HSA only at h = 0.03; 0.25; 0.41; 0.65). It is shown that the increase of both hydration degree above h greater than 0.1 and temperature above T greater than 200K leads to the appearance of intramolecular mobility in these proteins.