Structural transformations in protein crystals caused by controlled dehydration

Recent experiments in this laboratory on structural transformations caused by controlled dehydration of protein crystals have been reviewed. X-ray diffraction patterns of the following crystals have been examined under varying conditions of environmental humidity in the relative humidity range of 100-75%: a new crystal form of bovine pancreatic ribonuclease A grown from acetone solution in tris buffer (I), the well-known monoclinic form of the protein grown from aqueous ethanol (II), the same form grown from a solution of 2-methyl pentan-2,4-diol in phosphate buffer (III), tetragonal (IV), orthorhombic (V), monoclinic (VI) and triclinic (VII) hen egg white lysozyme, porcine 2 Zn insulin (VIII), porcine 4 Zn insulin (IX) and the crystals of concanavalin A(X). I, II, IV, V and VI undergo one or more transformations as evidenced by discontinuous changes in the unit cell dimensions, the diffraction pattern and the solvent content. Such water-mediated transformations do not appear to occur in the remaining crystals in the relative humidity range explored. The relative humidity at which the transformation occurs is reduced when 2-methyl pentan-2,4-diol is present in the mother liquor. The transformations are affected by the crystal structure but not by the amount of solvent in the crystals. The X-ray investigations reviewed here and other related investigations emphasize the probable importance of water-mediated transformations in exploring hydration of proteins and conformational transitions in them.     

NMR study of the structural transition in the DNA-bound water system in the interval of physiological temperatures

Temperature dependence of spin-spin proton relaxation times of DNA and bound water and the content of bound water in the samples of DNA, saturated with water in the atmosphere with different relative humidities from 0 to 100% were studied by means of pulsed NMR. It is shown that the temperature transition in the system of DNA-bound water in the interval 18-35 degrees is observed only when the relative humidity is more than 70% and the double-stranded structure of DNA exists. The transition of DNA from one conformation into another passes through some intermediate state more labile and probably less ordered. This transition is accompanied by changes in the structure of the hydration shell. In the case when relative humidity is greater than 80%, the partial dehydration of DNA stimulated by the transition is observed. This dehydration increases with the increase of relative humidity.     

Analysis of thermal hysteresis protein hydration using the random network model

The hydration of polar and apolar groups can be explained quantitatively, via the random network model of water, in terms of differential distortions in first hydration shell water-water hydrogen bonding angle. This method of analyzing solute induced structural distortions of water is applied to study the ice-binding type III thermal hysteresis protein. The analysis reveals subtle but significant differences in solvent structuring of the ice-binding surface, compared to non-ice binding protein surface. The major differences in hydration in the ice-binding region are (i). polar groups have a very apolar-like hydration. (ii). there is more uniform hydration structure. Overall, this surface strongly enhances the tetrahedral, or ice-like, hydration within the primary hydration shell. It is concluded that these two specific features of the hydration structure are important for this surface to recognize, and preferentially interact with nascent ice crystals forming in liquid water.     

Molecular dynamics simulation of solvated protein at high pressure.

We have completed a molecular dynamics simulation of protein (bovine pancreatic trypsin inhibitor, BPTI) in solution at high pressure (10 kbar). The structural and energetic effects of the application of high pressure to solvated protein are analyzed by comparing the results of the high-pressure simulation with a corresponding simulation at low pressure. The volume of the simulation cell containing one protein molecule plus 2943 water molecules decreases by 24.7% at high pressure. This corresponds to a compressibility for the protein solution of beta = 1.8 x 10(-2) kbar-1. The compressibility of the protein is estimated to be about one-tenth that of bulk water, while the protein hydration layer water is found to have a greater compressibility as compared to the bulk, especially for water associated with hydrophobic groups. The radius of gyration of BPTI decreases by 2% and there is a one third decrease in the protein backbone atomic fluctuations at high pressure. We have analyzed pressure effects on the hydration energy of the protein. The total hydration energy is slightly (4%) more favorable at high pressure even though the surface accessibility of the protein has decreased by a corresponding amount. Large pressure-induced changes in the structure of the hydration shell are observed. Overall, the solvation shell waters appear more ordered at high pressure; the pressure-induced ordering is greatest for nonpolar surface groups. We do not observe evidence of pressure-induced unfolding of the protein over the 100-ps duration of the high-pressure simulation. This is consistent with the results of high-pressure optical experiments on BPTI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of various humidity on local dynamic structure of lysozyme in a spin-labeled tetragonal crystal

The dynamics of the side groups of amino acid residues and local conformational changes in the lysozyme molecule upon dehydration and rehydration of lysozyme crystals were studied by the methods of spin label, X-ray diffraction, and molecular dynamics. The His15 residue of lysozyme from chicken egg white was modified by spin label, and spin-labeled tetragonal crystals of the protein were grown. The spatial structure of the covalently bound spin label and its immediate surroundings in the lysozyme tetragonal crystal was determined. The conformation of a fragment of the lysozyme molecule with the spin label on His15, optimized by the method of molecular dynamics, closely agreed with X-ray data. It was found by the X-ray diffraction analysis that a decrease in relative humidity to 40% is accompanied by both a decrease in the unit cell volume by 27% and a change in the diffraction field of roentgenograms from 0.23 to 0.60 HM. The dehydration of spin-labeled lysozyme crystals leads to an anomalous widening of EPR peaks without changes in their position. The dehydration in the humidity range studied has a two-stage character. The decrease in humidity to 75% is accompanied by a sharp change in the parameters measured, and on further decrease in humidity to 40% they change insignificantly. The first stage is caused by the removal of the greater part of molecules of bulk water, and the second stage is due to the removal of the remaining bulk water and possible changes in the dynamics of weakly bound water molecules and their position. The simulation of experimental EPR spectra showed that the anomalous broadening of the spectrum upon dehydration is related to an increase in the dispersion of spin label orientations induced by changes in the network of hydrogen bonds generated by water molecules in the vicinity of the spin label and a possible turn (by no more than 5 degrees) of the entire protein molecule. After rehydration, the physical state of the lysozyme crystal did not return to the starting point.     

Solvent structure in crystals of trypsin determined by X-ray and neutron diffraction.

The solvent structure in orthorhombic crystals of bovine trypsin has been independently determined by X-ray diffraction to 1.35 A resolution and by neutron diffraction to 2.1 A resolution. A consensus model of the water molecule positions was obtained using oxygen positions identified in the electron density map determined by X-ray diffraction, which were verified by comparison to D2O-H2O difference neutron scattering density. Six of 184 water molecules in the X-ray structure, all with B-factors greater than 50 A2, were found to be spurious after comparison with neutron results. Roughly two-thirds of the water of hydration expected from thermodynamic data for proteins was localized by neutron diffraction; approximately one-half of the water of hydration was located by X-ray diffraction. Polar regions of the protein are well hydrated, and significant D2O-H2O difference density is seen for a small number of water molecules in a second shell of hydration. Hydrogen bond lengths and angles calculated from unconstrained refinement of water positions are distributed about values typically seen in small molecule structures. Solvent models found in seven other bovine trypsin and trypsinogen and rat trypsin structures determined by X-ray diffraction were compared. Internal water molecules are well conserved in all trypsin structures including anionic rat trypsin, which is 65% homologous to bovine trypsin. Of the 22 conserved waters in trypsin, 19 were also found in trypsinogen, suggesting that they are located in regions of the apoprotein that are structurally conserved in the transition to the mature protein. Seven waters were displaced upon activation of trypsinogen. Water structure at crystal contacts is not generally conserved in different crystal forms. Three groups of integral structural water molecules are highly conserved in all solvent structures, including a spline of water molecules inserted between two beta-strands, which may resemble an intermediate in the formation of beta sheets during the folding of a protein.     

Structure of d(CACGTG), a Z-DNA hexamer containing AT base pairs.

The left-handed Z-DNA conformation has been observed in crystals made from the self-complementary DNA hexamer d(CACGTG). This is the first time that a non disordered Z form is found in the crystal structure of an alternating sequence containing AT base pairs without methylated or brominated cytosines. The structure has been determined and refined to an agreement factor R = 22.9% using 746 reflections in the resolution in the resolution shell 7 to 2.5 A. The overall shape of the molecule is very similar to the Z-structure of the related hexamer d(CG)3 confirming the rigidity of the Z form. No solvent molecules were detected in the minor groove of the helix near the A bases. The disruption of the spine of hydration in the AT step appears to be a general fact in the Z form in contrast with the B form. The biological relevance of the structure in relation to the CA genome repeats is discussed.     

Molecular level probing of preferential hydration and its modulation by osmolytes through the use of pyranine complexed to hemoglobin

Two spectroscopic probes are used to expose molecular level changes in hydration shell water interactions that directly relate to such issues as preferential hydration and protein stability. The major focus of the present study is on the use of pyranine (HPT) fluorescence to probe as a function of added osmolytes (PEG, urea, trehalose, and magnesium), the extent to which glycerol is preferentially excluded from the hydration shell of free HPT and HPT localized in the diphosphoglycerate (DPG) binding site of hemoglobin in both solution and in Sol-Gel matrices. The pyranine study is complemented by the use of vibronic side band luminescence from the gadolinium cation that directly exposes the changes in hydrogen bonding between first and second shell waters as a function of added osmolytes. Together the results form the basis for a water partitioning model that can account for both preferential hydration and water/osmolyte-mediated conformational changes in protein structure.     

Dependence of the hydration shell structure in the minor groove of the DNA double helix on the groove width as revealed by Monte Carlo simulation.

The hydration shell of several conformations of the polynucleotides poly(dA).poly(dT), poly(dA).poly(dU), and poly(dA-dI).poly(dT-dC) has been simulated using the Monte Carlo method (Metropolis sampling). Calculations have shown that the structure of the hydration shell of the minor groove greatly depends on its width. In conformations with a narrowed minor groove, the first layer of the hydration shell of this groove has only one molecule per nucleotide pair that forms H bonds with purine N3 of one pair and pyrimidine O2 of the next pair. The second layer of the hydration shell of such conformations contains molecules that form H bonds between two adjacent molecules of the first layer. The probability of formation of hydration spine is about 20% while the bridges of the first layer are formed with a probability of about 70%. In the first layer of the minor groove of the B-DNA conformation with wide minor groove there are approximately two water molecules per base pair that form H bonds with purine N3 or pyrimidine O2 and with the sugar ring oxygen of the adjacent nucleotide. The probability of simultaneous H bonding of a water molecule with N3 (or O2) and O of sugar ring is about 30%. The results of simulation suggest that hydration spine proposed for the narrowed minor groove of oligonucleotide crystals [H. R. Drew, and R. E. Dickerson (1981) Journal of Molecular Biology, Vol. 151, pp. 535-556] can be formed in fibers of poly(dA).poly(dT), poly(dA).poly(dU), and poly(dA-dI).poly(dT-dC) as well as in DNA fragments of these sequences in solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydration of B-DNA: comparison between the water network around poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) on the basis of Monte Carlo computations

A computational method is elaborated for studying the water environment around regular polynucleotide duplexes; it allows rigorous structural information on the hydration shell of DNA to be obtained. The crucial aspect of this Monte Carlo simulation is the use of periodical boundary conditions. The output data consists of local maxima of water density in the space near the DNA molecule and the properties of one- and two-membered water bridges as function of pairs of polar groups of DNA. In the present paper the results for poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) are presented. The differences in their hydration shells are of a purely structural nature and are caused by the symmetry of the polar groups of the polymers under study, the symmetry being reflected by the hydration shell. The homopolymer duplex hydration shell mirrors the mononucleotide repeat. The water molecules contacting the polynucleotide in the minor groove are located nearly in the plane midway between the planes of successive base pairs. One water molecule per base pair forms a water bridge facing two polar groups of bases from adjacent base pairs and on different strands making a "spine"-like structure. In contrast, the major groove hydration is stabilized exclusively by two-membered water bridges; the water molecules deepest in the groove are concentrated near the plane of the corresponding base pair. The alternating polymer is characterized by a marked dyad symmetry of the hydration shell corresponding to the axis between two successive base pairs. The minor groove hydration of the dCpdG step resembles the characteristic features of the homopolymer, but the bridge between the O2 oxygens of the other base-stacking type is formed by two water molecules. The major groove hydration is characterized by high probability of one-membered water bridges and by localization of a water molecule on the dyad axis of the dGpdC step. The found structural elements are discussed as reasonable invariants of a dynamic hydration shell.     

Structure and mechanism of HpcG, a hydratase in the homoprotocatechuate degradation pathway of Escherichia coli

HpcG catalyses the hydration of a carbon-carbon double bond without the aid of any cofactor other than a simple divalent metal ion such as Mg(2+). Since the substrate has a nearby carbonyl group, it is believed that it first isomerises to form a pair of conjugated double bonds in the enol tautomer before Michael addition of water. Previous chemical studies of the reaction, and that of the related enzyme MhpD, have failed to provide a clear picture of the mechanism. The substrate itself is unstable, preventing co-crystallisation or soaking of crystals, but oxalate is a strong competitive inhibitor. We have solved the crystal structure of the protein in the apo form, and with magnesium and oxalate bound. Modelling substrate into the active site suggests the attacking water molecule is not part of the metal coordination shell, in contrast to a previous proposal. Our model suggests that geometrically strained cis isomer intermediates do not lie on the reaction pathway, and that separate groups are involved in the isomerisation and hydration steps.     

Deuterium solid-state NMR investigations of exchange labeled oriented purple membranes at different hydration levels

Oriented purple membranes were equilibrated under controlled (2)H(2)O relative humidity ranging from 15% to 93% and introduced into the magnetic field of an NMR spectrometer with the membrane normal parallel to the magnetic field direction. Deuterium solid-state NMR spectra of these samples resolved four deuteron populations. Deuterons that have exchanged with amide protons of the protein exhibited a broad spectral line shape (<150 kHz). Furthermore, a broadened signal of deuterons tightly associated with protein and lipid is detected at low hydration, as well as two additional water populations that were present when the samples were equilibrated at >/=75% relative humidity. These latter ones are characterized by narrow quadrupolar splittings (<2.5 kHz) and orientation-dependent chemical shifts. Their deuterium relaxation times, measured as a function of temperature, indicate correlation times in the fast regime (10(-10) s) and activation energies of 13 kJ/mol (at 86% relative humidity). Differences in T(1) and T(2) relaxation together with small residual quadrupole splittings show that the mobility of the deuterons is anisotropic. The occurrence of these mobile water populations at high levels of purple membrane hydration (>/=75% relative humidity) correlate with proton pumping activity of bacteriorhodopsin, the fast kinetics of M-decay in the bacteriorhodopsin photocycle, and structural alterations of the protein during the M-state, which have been described previously.     

Structure of DNA hydration shells studied by Raman spectroscopy

We have used Raman scattering to study the water O-H stretching modes at approximately 3450 and approximately 3220 cm-1 in DNA films as a function of relative humidity (r.h.). The intensity of the 3220-cm-1 band vanishes as the r.h. is decreased from 98% to around 80%, which indicates that the hydrogen-bond network of water is disrupted in the primary hydration shell (which therefore cannot have an "ice-like" structure). The number of water molecules in the primary hydration shell was determined from the intensity of the approximately 3200-cm-1 band as about 30 water molecules per nucleotide pair. The approximately 3400-cm-1 O-H stretch band was used for determining the total water content, and this band persists at 0% r.h., implying that 5-6 tightly bound water molecules per nucleotide pair remain. The frequency of the approximately 3400-cm-1 O-H stretch mode is lower by 30 to 45 cm-1 in the primary hydration shell compared to free water. The water content as a function of r.h. obtained from these experiments agrees with gravimetric measurements. The disappearance of the approximately 3200-cm-1 band and the shift of the approximately 3400-cm-1 O-H stretch band provide a reliable way of measuring the hydration number of DNA.     

Hydration of biological molecules: lipids versus nucleic acids

We used FTIR spectroscopy to comparatively study the hydration of films prepared from nucleic acids (DNA and double-stranded RNA) and lipids (phosphatidylcholines and phosphatidylethanolamines chosen as the most abundant ones) at room temperature by varying the ambient relative humidity in terms of solvent-induced structural changes. The nucleic acids and phospholipids both display examples of polymorphism on the one hand and structural conservatism on the other; even closely related representatives behave differently in this respect. DNA undergoes a hydration-driven A-B conformational transition, but RNA maintains an A-like structure independently of the water activity. Similarly, a main transition between the solid and liquid-crystalline phases can be induced lyotropically in certain phosphatidylcholines, while their phosphatidylethanolamine counterparts do not exhibit chain melting under the same conditions. A principal difference concerning the structural changes that occur in the studied biomolecules is given by the relevant water-substrate stoichiometries. These are rather high in DNA and often low in phospholipids, suggesting different mechanisms of action of the hydration water that appears to induce structural changes on global- and local-mode levels, respectively.     

Role of hydration water in protein unfolding

In this paper, following our work on the two-state outer neighbor mixed bonding model of water, it is proposed that polar groups promote the formation of the low density ice Ih-type bonding in their neighborhood, whereas nonpolar groups tend to promote the higher density ice II-type structure. In a protein, because of the large numbers of exposed polar and nonpolar groups, large changes in the neighboring water structure can occur. These changes, of course, depend on whether the protein is in its native or its unfolded state and will be shown here to have a direct impact on the thermodynamics of protein unfolding at both high and low temperatures. For example, it is known that the polar hydration entropies become rapidly more negative with increasing temperature. This very unusual behavior can be directly related to the promotion in the outer bulk liquid of the more stable Ih-type bonding at the expense of II-type bonding by polar groups of the protein. In contrast, nonpolar groups have an opposite effect on the thermodynamics. It is the delicate balance created by these outer hydration contributions, mixed with ordinary thermodynamic contributions from the inner hydration shell and those from hydrogen-bond and van der Waals forces within the protein molecule itself that is responsible for both heat and cold denaturation of proteins.     

X-ray structural studies of Mycobacterium tuberculosis RRF and a comparative study of RRFs of known structure. Molecular plasticity and biological implications

The crystal structure of Mycobacterium tuberculosis ribosome recycling factor has been determined and refined against three X-ray diffraction data sets, two collected at room temperature and the other at 100K. The two room-temperature data sets differ in the radiation damage suffered by the crystals before the data used for processing were collected. A comparison between the structures refined against the two data sets indicates the possibility of radiation-induced conformational change. The L-shaped molecule is composed of a long three-helix bundle domain (domain I) and a globular domain (domain II) connected by a linker region. The main difference between the room-temperature structure and the low temperature structure is in the rotation of domain II about an axis close to its libration axis. This observation and a detailed comparative study of ribosome recycling factors (RRFs) of known structures led to an elaboration of the present understanding of the structural variability of RRF. The variability involves a change in the angle between the two arms of the molecule, a rotation of domain II in a plane nearly perpendicular to the axis of the helix bundle and an internal rotation of domain II. Furthermore, the domains and the linker could be delineated into fixed and variable regions in a physically meaningful manner. The relative mobility of the domains of the molecule in the crystal structure appears to be similar to that in the ribosome--RRF complex. That permits a meaningful discussion of the structural features of RRF in terms of ribosome--RRF interactions. The structure also provides insights into the results of inter-species complementation studies.     

高分辨率高精度蛋白质晶体结构

自从六十年代初期应用X射线晶体衍射方法测定第一个蛋白质(肌红蛋白)的三维结构以来,我们关于蛋白质三维结构的知识已有了相当的积累.截止86年7月的统计,已有286个蛋白质分子的原子坐标存入国际蛋白质数据库.以此为基础,一些重要生命活动的结构机理已经在三维水平上得到精细阐明(参见),一个由分子生物学和X射线晶体学结合形成的新领域——蛋白质晶体学,应运而生.二十多年来,这一领域的发展已经历了二个阶段.第一阶段大体从六十年代初到七十年代中期,在这期间,分析方法和技术从突破发展到成熟和实用,并有近40个蛋白质结构测定出来,使我们获得了蛋白质结构知识的面面观.     

Experimental study of DNA hydratation

The present article as a review of experimental investigations of water-DNA interaction in solutions, films, fibres, and crystals. The discussion of the experimental data is directed to the analysis of the sizes of the hydration shells of the DNA atomic groups, the distances at which atomic groups of DNA affect the hydration of each other; the structural, thermodynamic and kinetic characteristics of the water in the DNA hydration shell and their relations with DNA structure. The modern views on the mechanisms by means of which the water affects the physico-chemical properties of DNA are discussed.     

An asymmetric dimer of beta-lactoglobulin in a low humidity crystal form--structural changes that accompany partial dehydration and protein action

Dimeric lactoglobulin molecules exist in the open conformation at basic pH, whereas they exist in the closed conformation at acidic pH, after undergoing the Tanford transition around neutral pH. Orthorhombic crystals consisting of molecules in the open conformation, grown close to neutral pH, undergo a water-mediated transformation when the relative humidity around the crystals is reduced. The two subunits in the dimer are related by a crystallographic twofold axis in the native crystals while the dimer is asymmetric in the low humidity form. Interestingly, one of the subunits in the dimer in the low humidity form is in an open conformation while the other is in a closed conformation. This is the first observation of such an asymmetric dimer. A hydrogen bond between the side chains of Gln35 and Tyr42 exists and the side chain of Glu89 is substantially buried in the closed subunit of the asymmetric unit, as in other structures with molecules in the closed conformation. However, the closure of the EF loop is not complete; its conformation can be described as half-closed. A comparison of different crystal structures of beta-lactoglobulin indicates that the conformation of the loops in the molecule is substantially influenced by other factors such as crystal packing, the pH, and the composition of the medium, while the change in the conformation of the EF loop follows the Tanford transition. The mutual disposition of the two subunits in the low humidity form is halfway between those in the open and closed structures. The present work further demonstrates that structural changes that occur during partial dehydration could mimic those that occur during the action of proteins.