Packing analysis of carbohydrates and polysaccharides. IV. A new method for detailed crystal structure refinement of polysaccharides and its application to V-amylose

A method is described for predicting and solving crystal structures of linear homopolysaccharides. The method is based on the refinement of the structure with respect to either stereochemical constraints or x-ray diffraction intensities. In the refinement process, all conformational and packing features of the molecule, such as bond lengths, bond angles, conformational angles, nonbonded contacts, hydrogen bonds, etc., can be allowed to vary until the structure reaches both a conformation and crystalline packing that are in minimum disagreement with the stereochemical restraints and the diffraction data. In this fashion, both packing and conformational features of the structure can be simultaneously refined, and not separately as has been the custom in the past. The refinement procedure is based on a method of constrained optimization which possesses improved characteristics of reaching a solution and avoiding false minima, in comparison with least squares methods. The procedure is, in addition, capable of easily finding molecules of solvent of crystallization. The method was applied to further refining the previously solved crystal structure of V-amylose. The results indicated that contrary to the previously found six-fold molecular symmetry in the P2₁2₁2₁ space group, the V-amylose molecule exhibits only two-fold symmetry with the asymmetric unit consisting of three glucose residues in one-half turn of the helix. The three residues are nonequivalent principally due to unequal rotational positions of the hydroxymethyl groups. The crystal structure of V-amylose predicted from stereochemical refinement was identical in all details with that obtained from refining against X-ray data. The excellent agreement with the diffraction data was indicated by the crystallographic disagreement index R = 0.25.     

Molecular dynamics of oligopeptides. 3. Maps of levels of free energy of modified dipeptides and dynamic correlation in amino acid residues]

A method of free energy maps for studying the dynamic correlations of fluctuations in molecules with conformational mobility is proposed. An agreement between the structure of the free energy level map and the type of the corresponding cross-correlation function (in the presence and absence of the correlation of fluctuations in conformational freedom degree in modified dipeptides) was established.     

Effect of sodium ion concentration on transfer RNA conformation in solution studied by Rayleigh light scattering

The sodium ion concentration dependent conformational changes of transfer RNA (unfractionated tRNA from baker's yeast) have been studied in unbuffered aqueous solutions by Rayleigh light scattering. Changes of the optical parameters of the molecule indicated the following conformational changes of tRNA with increasing NaCl concentration: in salt-free solution tRNA molecules have an irregular hairpin loop-like structure in which the orientation of base rings is not correlated. Upon addition of a small amount of NaCl (0.005 M) an increasing ordering of this structure is observed. In 0.1 M-NaCl the molecule has an extended structure with ordered regions (arms). Further increase of sodium ion concentration up to 2 M results in folding of the extended structure and formation of a compact and rigid conformation in which most of the bases are nearly perpendicular to the symmetry axis of the molecule.     

Mass-weighted molecular dynamics simulation of cyclic polypeptides.

A modified molecular dynamics (MD) method in which atomic masses are weighted was developed previously for studying the conformational flexibility of neuroregulating tetrapeptide Phe-Met-Arg-Phe-amide (FMRF-amide). The method has now been applied to longer and constrained molecules, namely a disulfide-linked cyclic hexapeptide, c[CYFQNC], and its linear and "pseudo-cyclic" analogues. The sampling of dehedral conformational space of teh linear hexapeptide in mass-weighted MD simulations was found to be improved significantly over conventional MD simulations, as in the case of the shorter FMRF-amide molecule studied previously. In the cyclic hexapeptide, the internal constraint of the molecule due to the intramolecular disulfide bond (hence the absence of free terminals in the molecule) does not adversely affect the significant improvement of conformational sampling in mass-weighted MD simulations over normal MD simulations. The pseudo-cyclic polypeptide is identical to the linear CYFQNC molecule in amino acid sequence (i.e., side chains of the cysteine residues are reduced), but the positions of its two terminal heavy atoms were held fixed in space such that the molecule has a nearly cyclic conformation. For this molecule, the mass-weighted MD simulation generated a wide range of polypeptide backbone conformations covering the internal dihedral degrees of freedom; moreover, the physical space of the pseudo-cyclic structure was also sampled in a complete revolution of the entire molecular fragment about the two fixed termini during the simulation. These characteristics suggest that mass-weighted MD can also be an extremely useful method for conformational analyses of constrained molecules and, in particular, for modeling loops on protein surfaces.     

Solvent accessibilities in glycyl, alanyl and seryl dipeptides.

Theoretical studies on glycyl-alanyl and seryl dipeptides were performed to determine the probable backbone and side-group conformations that are preferred for solvent interaction. By following the method of Lee & Richards [(1971) J. Mol. Biol. 55, 379-400], a solute molecule is represented by a set of interlocking spheres of appropriate van der Waals radii assigned to each atom, and a solvent (water) molecule is rolled along the envelope of the van der Waals surface, and the surface accessible to the solvent molecule, and hence the solvent accessibility for a particular conformation of the solute molecule, is computed. From the calculated solvent accessibilities for various conformations, solvation maps for dipeptides were constructed. These solvation maps suggest that the backbone polar atoms could interact with solvent molecules selectively, depending on the backbone conformation. A conformation in the right-handed bridge (zetaR) region is favoured for both solvent interaction and intrachain hydrogen-bonding. Also the backbone side-chain hydrogen-bonding within the same dipeptide fragment in proteins is less favoured than hydrogen-bonding between side chain and water and between side chain and atoms of other residues. Solvent accessibilities suggest that very short distorted alphaR-helical and extended-structural parts may be stabilized via solvent interaction, and this could easily be possible at the surface of the protein molecules, in agreement with protein-crystal data.     

Conformational properties of Pro-Pro sequences. I. Crystal structures of two dipeptides with L-Pro-L-Pro and L-Pro-D-Pro sequences

The crystal structures of Bu^tCO-L -Pro-L -Pro-NHMe, H₂O (1: monoclinic; P2₁; a = 6.662, b = 11.067, c = 12.205 Å; β = 96.28°) and Bu^tCO-L -Pro-D -Pro-NHMe (2: monoclinic; P2₁; a = 10.770, b = 15.039, c = 11.325 Å; β = 110.00°) have been solved by x-ray diffraction. Structure 1 accommodates an open disposition with intermolecular interactions involving the water molecule, while 2 is βII-folded by an intramolecular i + 3 → i hydrogen bond. In both derivatives, small thermal parameters are indicative of fairly fixed conformations for the proline rings. Comparison between conformations of either isolated or adjacent L -Pro residues in the crystal structures of unstrained oligopeptides shows that the conformational properties of L -Pro-L -Pro sequences are probably a simple combination of those found for isolated L -Pro residues.     

Mass-weighted molecular dynamics simulation and conformational analysis of polypeptide.

Atomic motions in protein molecules have been studied by molecular dynamics (MD) simulations; dynamics simulation methods have also been employed in conformational studies of polypeptide molecules. It was found that when atomic masses are weighted, the molecular dynamics method can significantly increase the sampling of dihedral conformation space in such studies, compared to a conventional MD simulation of the same total simulation time length. Herein the theoretical study of molecular conformation sampling by the molecular dynamics-based simulation method in which atomic masses are weighted is reported in detail; moreover, a numerical scheme for analyzing the extensive conformational sampling in the simulation of a tetrapeptide amide molecule is presented. From numerical analyses of the mass-weighted molecular dynamics trajectories of backbone dihedral angles, low-resolution structures covering the entire backbone dihedral conformation space of the molecule were determined, and the distribution of rotationally stable conformations in this space were analyzed quantitatively. The theoretical analyses based on the computer simulation and numerical analytical methods suggest that distinctive regimes in the conformational space of the peptide molecule can be identified.     

The crystal struture of monoclinic ribonuclease-S at six angstroms resolution.

An independent structure analysis has been made of ribonuclease-S crystallized in a monoclinic space group C2 at 6 Å resolution. The conformations of the two crystallographically independent molecules (molecule ZA and ZB) were compared with that of a chemically identical molecule (molecule Y) crystallized in a trigonal space group P3₁21, the structure of which has been solved to 2.0 Å resolution by Wyckoff et al. (1970). The N-terminal tail of the S-protein of molecule ZA assumes a unique conformation somewhat resembling that of ribonuclease-A, while the corresponding part of molecule ZB assumes about a similar conformation to that of molecule Y. Apart from the solvated terminal region, the overall arrangements of various features of the three structures are very similar, although possibilities of local conformational differences are not considered at this stage of the analysis. The environments of the three molecules in the crystal lattice are compared in detail. Two of the four molecules in the primitive cell are related to each other by a crystallographic 2-fold axis very similar to a 2-fold relationship found in the Y-form. All other relationships are quite different.     

Conformational investigation of alpha, beta-dehydropeptides. XI. Molecular and crystal structure of Ac-(Z)-deltaPhe-NMe2 as compared to those of related molecules.

A series of three homologous dimethyldiamides Ac-(Z)-deltaPhe-NMe2, Ac-L-Phe-NMe2 and Ac-DL-Phe-NMe2 have been synthesized and their structures determined from single-crystal X-ray diffraction data. To learn more about the conformational preferences of the compounds studied, the fully relaxed phi, psi conformational energy maps on the free molecules of Ac-deltaAla-NMe2 and Ac-(Z)-deltaPhe-NMe2 were obtained with the HF/3-21G method and the calculated minima re-optimized with the DFT/B3LYP/6-31G** method. The crystal state results have been compared with the literature data. The studied dimethyldiamide Ac-deltaXaa-NMe2 combines the double bond in positions alpha, beta and the C-terminal tertiary amide within one molecule. As the representative probe with deltaXaa = deltaAla, (Z)-deltaLeu and (Z)-deltaPhe shows, in the solid state they adopt the conservative conformation with phi, psi approximately -45 degrees, approximately 130 degrees and with a non-planar tertiary amide bond, whatever the packing forces are. This conformation is located on the Ramachandran map in region H/F, which is of high-energy for common amino acids, but not so readily accessible to them. The free molecule calculations on Ac-deltaAla-NMe2 and Ac-(Z)-deltaPhe-NMe2 reveal that, in spite of dissimilar overall conformational profiles of these molecules, this structure is one of their low-energy conformers and for Ac-(Z)-deltaPhe-NMe2 it constitutes the global minimum. So, the theoretical results corroborate those experimental results proving that this structure is robust enough to avoid conformational distortion due to packing forces. In contrast to Ac-deltaXaa-NMe2, the saturated Ac-L/DL-Xaa-NMe2 shows the constancy of the associative patterns but do not prefer any molecular structure in the solid state.     

The importance of dynamic light scattering in obtaining multiple crystal forms of Trypanosoma brucei PGK.

Phosphoglycerate kinase (PGK) catalyzes the phosphoryl transfer between 1,3 bis-phosphoglycerate and ADP to form 3-phosphoglycerate and ATP, undergoing significant conformational changes during catalysis. To more precisely document this reaction and the corresponding conformational changes, we have crystallized Trypanosoma brucei PGK in several crystal forms: (1) in the presence of 3-phosphoglycerate and MgADP, PGK crystallizes with four molecules in the asymmetric unit; (2) in the presence of the ATP analog, AMP-PNP, PGK crystallizes in a similar form; (3) in the presence of the bisubstrate analog, adenylyl 1,1,5,5-tetrafluoropentane-1,5-bisphosphonate, PGK crystals grow with one molecule in the asymmetric unit. Large scale expression and purification of T. brucei PGK from an E. coli overexpression system was required to obtain sufficient enzyme yields. Results from dynamic light scattering experiments allowed us to identify substrates and analogs which were amenable for crystallization. Ease of crystal growth and diffraction quality for a particular PGK-ligand complex is highly consistent with the apparent monodispersity of the complex in solution as judged by dynamic light scattering. The three-dimensional structures of the various enzyme-ligand complexes are currently being exploited to obtain a better understanding of PGK catalysis, as well as for structure based design of enzyme inhibitors to be used in the development of anti-trypanosomal agents.     

Topography of cyclodextrin inclusion complexes : Part II. The iodine-cyclohexa-amylose tetrahydrate complex; its molecular geometry and cage-type crystal structure

Iodine-cyclohexa-amylose tetrahydrate [(C₆H₁₀O₅)₆ ·I₂·d4H₂O] crystallizes in the orthorhombic space-group P2₁2₁2₁, a  14.240 Å, b  36.014 Å, c  9.558 Å. The structure was solved by heavy-atom techniques and refined by least-squares methods to a conventional discrepancy index R  0.148 for the 2872 observed data. The six d-glucose residues are in the C1 chair conformation; the conformational angles vary in magnitude from 45 to 66°, the angles O(5)-C(5)-C(6)-O(6) are close to · 70°, and the six O(4) atoms are almost coplanar (r.m. s. displacement 0.13 Å). Only four of the six O(2) ?O(3) intramolecular hydrogen bonds have formed, which renders the molecule less symmetrical and more conical-shaped than in the previously determined α-cyclodextrin-potassium acetate complex. The iodine molecule is coaxial with the cyclohexa-amylose molecule. The I-I distance is a conventional 2.677 Å. Close interactions between the iodine atoms and the host molecule comprise carbon atoms C(5) and C(6) and oxygen atoms O(4), with interatomic distances all equal to or greater than van der Waals contacts. Intermolecular, almost-linear, short contacts O ? I-I?O with I?O distances of 3.22 and 3.07 Å indicate attractive interaction.The molecules are arranged in herring-bone “cage-type” fashion, with the four water molecules as space-filling mediators; the structure is held together by an intricate network of hydrogen bonds.     

Toward a general mixed quantum/classical method for the calculation of the vibronic ECD of a flexible dye molecule with different stable conformers: Revisiting the case of 2,2,2‐trifluoro‐anthrylethanol

We extend a recently proposed mixed quantum/classical method for computing the vibronic electronic circular dichroism (ECD) spectrum of molecules with different conformers, to cases where more than one hindered rotation is present. The method generalizes the standard procedure, based on the simple Boltzmann average of the vibronic spectra of the stable conformers, and includes the contribution of structures that sample all the accessible conformational space. It is applied to the simulation of the ECD spectrum of (S)‐2,2,2‐trifluoroanthrylethanol, a molecule with easily interconvertible conformers, whose spectrum exhibits a pattern of alternating positive and negative vibronic peaks. Results are in very good agreement with experiment and show that spectra averaged over all the sampled conformational space can deviate significantly from the simple average of the contributions of the stable conformers. The present mixed quantum/classical method is able to capture the effect of the nonlinear dependence of the rotatory strength on the molecular structure and of the anharmonic couplings among the modes responsible for molecular flexibility. Despite its computational cost, the procedure is still affordable and promises to be useful in all cases where the ECD shape arises from a subtle balance between vibronic effects and conformational variety.     

Studying compaction-decompaction of DNA molecules induced by surfactants

The mechanism and detailed processes of DNA compaction and decompaction are essential for the life activities, as well as for the researches in the molecular biology, genetics and biomedicine. The compaction of two kinds of DNA molecules caused by Cetyltrimethyl Ammonium Bromide (CTAB) and their decompaction induced with sodium dodecyl sulfate (SDS) or excessive amount of CTAB have been investigated with multiple perspectives such as the UV-VIS spectrophotometry, dynamic light scattering, and zeta potential. The compaction phenomenon of DNA can easily be observed when the CTAB combines with the DNA, not just when the molar ratio Q_CTAB/Q_DNA is approximately equal to 1 as the conventional recognition, but also when Q_CTAB/Q_DNA <1,DNA can be compacted; Molecular state of DNA is only changed in the conformational structure, but not in the chemical structure. Finally, a model is suggested to help catch on the biophysical mechanism of DNA chain conformational change.     

Effects of hydration on scale factors for ab initio force constants. IV. trans and cis N-methylacetamide

Scaled quantum mechanical force fields have been calculated using the 4-31G basis for trans and cis conformers of both isolated and water-solvated N-methylacetamide (NMA). (1) A single set of scale factors for isolated NMA yields relatively correct predictions of the shifts in vibrational frequencies between the trans and cis conformers. This is also true of a single set of scale factors for trans and cis NMA in water. The total standard deviation between measured and calculated frequencies for trans NMA in both isolated and solvated states is 6 cm^?1. This implies that it should be possible to use a single set of scale factors to accurately predict the vibrational spectra of a peptide in a variety of conformational states. (2) The computationally predicted effect of hydration on force constants for the supermolecule NMA · nH₂O are generally consistent with the experimentally measured effects of hydration on scale factors. These results indicate that supermolecule calculations can be useful in predicting the effects of hydration on spectra. (3) Three types of scale factors are calculated as follows: (a) first from ab initio calculations on an isolated molecule using frequencies measured from isolated molecules; (b) second from calculations on an isolated molecule using frequencies measured from water-solvated or otherwise hydrogen-bonded molecules; (c) and third from supermolecule calculations on a molecule hydrogen-bonded to water, using frequencies measured from water-solvated molecules. (4) The third type of scale factors are similar to the first type, for confidently measured modes, even though some of the force constants are very different. This suggests that one set of scale factors may be transferable to both isolated and hydrogen-bonded molecules, and that the simple representation of hydration used here may be a useful approximation. The second type of scale factors yield accurate frequencies, but they may not be generally transferable.     

A Study of stepwise conformational changes in poly(β-benzyl-L-aspartate) with the helix–coil transition by means of proton and carbon-13 NMR

The helix–coil transition for poly(β-benzyl-L -aspartate) [poly(Asp[OBzl])] in solvent mixtures of trifluoroacetic acid/deuterated chloroform (F₃AcOH/CDCl₃) was studied by means of proton and carbon-13 nmr. Conformational fixation of the side chain occurs before the coil–helix transition of the backbone, when neighboring phenyl rings face each other. Another type of conformational fixation occurs in the side chain after the coil–helix transition of the backbone. These conformational changes of the side chain are due to the changes of the strength of the interaction between the side-chain ester group and the F₃AcOH molecule. In the absence of F₃AcOH (coil-forming solvent), the polymer has a rather rigid structure in which the side chain may wrap around the backbone. These conformational changes of the polymer are closely related to the changes of the interaction between the polymer and F₃AcOH molecules.     

Appearance of Neoantigen in Mouse IgG1 Upon Reduction of Interchain Disulfide Bridges: Assessment of Local and General Conformational Rearrangements Using Monoclonal Antibody and Small-angle X-ray Scattering

Abstract

Mouse hybridoma antibody E5D2 reacting with murine mono- and polyclonal IgG1 has been produced. MonAb E5D2 recognizes the antigenic determinant (epitope) buried in intact IgG1 and expressed upon mild reduction of interchain S-S bridges. Neither H nor L chains alone maintain epitope E5D2. Reassociation of gamma 1 chains (H chains of IgG1) with L chains results in complete restoration of this antigenic determinant. The data strongly suggest that epitope E5D2 depends on the quaternary structure of IgG1. The epitope is also expressed by reduced F(ab)₂ fragment of IgG1 but is not connected with its antigen binding site. The likely localization of the epitope E5D2 is the interface between C_H and C_L domains. The second produced monAb F6C2 reacts with C_H1-C_L region of reduced mouse IgG2.

Small-angle X-ray scattering experiments have demonstrated pronounced decrease of the radius of gyration of reduced IgG1 as compared to the intact one. This indicates general conformational changes of IgG1 molecule following mild reduction of Fab region S-S groups. Epitope E5D2 is the first quaternary antigenic subclass specific determinant described for C the region of mouse IgG. Thus, serologic expression of epitope E5D2 reveals precise conformational perturbations of small area near reduced S-S bridges while small-angle scattering demonstrates accompanying general transformation of IgG structure.     

The ternary 5-S RNA complex of proteins L 18 and L 25. A small-angle X-ray scattering titration study.

The 5-S RNA (A) and the proteins L 18 (B) and L25 (C) from Escherichia coli ribosomes form a ternary complex of the type ABC with a stepwise stability constant, log K111 approximately equal to 6.5. This is indicated from X-ray scattering titrations recorded at 21 degrees C in ribosomal reconstitutional buffer. When the ternary ABC complex forms there is only a limited change in the scattering curve compared to that of 5-S RNA, indicating that 5-S RNA does not undergo a major conformational change during the complex formation. The increase in the radius of gyration from 3.61 nm (5-S RNA) to 3.95 nm (ABC complex) as well as the experimental scattering curve can be explained by models where it is assumed that the elongated L 18 and L25 models are quite far from the electron density centre and where the protein molecules interact mainly with the minor arms of the supposed Y-shaped 5-S RNA molecule.     

X-ray structure of the curare alkaloid (+) - tubocurarine dibromide

X-ray structure determination of the compound (C₃₇H₄₂N₂O₆)²⁺. 2Br⁻. 4CH₃OH, confirms that (+) - tubocurarine is a monoquarternary salt and has established that the molecule adopts different conformations in crystals of the dibromide and dichloride salts. The crystal structure is stabilised by a number of hydrogen bonds involving the two free hydroxyl groups and the tertiary nitrogen of the tubocurarine molecule, the bromide ions and the solvent molecules. The absolute configuration of the molecule, determined by X-ray anomalous scattering, confirms the configuration assigned earlier by chemical studies.     

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.