Preferred conformations and flexibility of aminoacyl side chain of penicillins

Possible conformations of penicillin G; d and l isomers of ampicillin; α-amino-α-methyl-benzyl penicillins and 3- pyridyl methyl penicillin have been studied by an energy minimization procedure using empirical potential functions. The preferred conformations of these antibiotics have been correlated with their biological activity. The conformational requirement of the antibiotic to be active against Gram-positive and Gram-negative (β-lactamase-negative) bacterial strains seems to be the same. The reduced activity of penicillin G against Gram-negative bacteria has been attributed to its lower ability to permeate the outer membrane. The flexibility of the sidechains of these antibiotics is also shown to be important for the desired biological activity.     

Structural basis of DNA flexibility

It has been recently shown by us, on the basis of crystal structure database that the flexibility of B-DNA double helices depends significantly on their base sequence. Our model building studies further indicated that the existence of bifurcated cross-strand hydrogen bonds between successive base pairs is possibly the main factor behind the sequence directed DNA flexibility. These cross-strand hydrogen bonds are, of course, weaker than the usual Watson-Crick hydrogen bonds and their bond geometry is characterized by relatively larger bond lengths and smaller bond angles. We have tried to improve our model structures by incorporating non-planarity of the amino groups in DNA bases due to the presence of lone pair electrons at the nitrogen atoms. Energy minimization studies have been carried out by using different quantum chemical methods, whereby it is found that in all cases of N-H....O type cross-strand hydrogen bonds, the bond geometry improves significantly. In the cases of N-H....N type hydrogen bonds, however, no such consistent improvements can be noticed. Perhaps the true picture would emerge only if all the other interactions present in the DNA macromolecule could be appropriately taken into account.     

Variability of the canonical loop conformations in serine proteinases inhibitors and other proteins

Canonical loops of protein inhibitors of serine proteinases occur in proteins having completely different folds. In this article, conformations of the loops have been analyzed for inhibitors belonging to 10 structurally different families. Using deviation in C_α-C_α distances as a criterion for loop similarity, we found that the P3-P3′ segment defines most properly the length of the loop. When conformational differences among loops of individual inhibitors were compared using root mean square deviation (rmsd) in atomic coordinates for all main chain atoms (Δr method) and rmsd operating in main chain torsion angles (Δt method), differences of up to 2.1 Å and 72.3°, respectively, were observed. Such large values indicate significant conformational differences among individual loops. Nevertheless, the overall geometry of the inhibitor-proteinase interaction is very well preserved, as judged from the similarity of C_α-C_α distances between C_α of catalytic Ser and C_α of P3-P3′ residues in various enzyme-inhibitor complexes. The mode of interaction is very well preserved both in the chymotrypsin and subtilisin families, as distances calculated for subtilisin-inhibitor complexes are almost always within the range of those for chymotrypsin-inhibitor complexes. Complex formation leads to conformational changes of up to 160° for χ₁ angle. Side chains of residue P1 and P2′ adopt in different complexes a similar orientation (χ₁ angle = −60° and −180°, respectively). To check whether the canonical conformation can be found among non–proteinase-inhibitor Brookhaven Protein Data Bank structures, two selection criteria—the allowed main chain dihedral angles and C_α-C_α distances for the P3-P3′ segment—were applied to all these structures. This procedure detected 132 unique hexapeptide segments in 121 structurally and functionally unrelated proteins. Partial preferences for certain amino acids occurring at particular positions in these hexapeptides could be noted. Further restriction of this set to hexapeptides with a highly exposed P1 residue side chain resulted in 40 segments. The possibility of complexes formation between these segments and serine proteinases was ruled out in molecular modeling due to steric clashes. Several structural features that determine the canonical conformation of the loop both in inhibitors and in other proteins can be distinguished. They include main chain hydrogen bonds both within the P3-P3′ segment and with the scaffold region, P3-P4 and P3′-P4′ hydrophobic interactions, and finally either hydrophobic or polar interactions involving the P1′ residue. Proteins 32:459–474, 1998. © 1998 Wiley-Liss, Inc.     

Nanomechanics of hemichannel conformations: connexin flexibility underlying channel opening and closing

Gap junctional hemichannels mediate cell-extracellular communication. A hemichannel is made of six connexin (Cx) subunits; each connexin has four transmembrane domains, two extracellular loops, and cytoplasmic amino- and carboxyl-terminals (CTs). The extracellular domains are arranged differently at non-junctional and junctional (gap junction) regions, although very little is known about their flexibility and conformational energetics. The cytoplasmic tail differs considerably in the size and amino acid sequence for different connexins and is predicted to be involved in the channel open and closed conformations. For large connexins, such as Cx43, the CT makes large cytoplasmic fuzz visible under electron microscopy. If this CT domain controls channel permeability by physical occlusion of the pore mouth, movement of this portion could open or close the channel. We used atomic force microscopy-based single molecule spectroscopy with antibody-modified atomic force microscopy tips and connexin mimetic peptide modified tips to examine the flexibility of extracellular loop and CT domains and to estimate the energetics of their movements. Antibody to the CT portion closer to the membrane stretches the tail to a shorter length, and the antibody to CT tail stretches the tail to a longer length. The stretch length and the energy required for stretching the various portions of the carboxyl tail support the ball and chain model for hemichannel conformational changes.     

Structural determinants of the conformations of medium-sized loops in proteins

Loops are integral components of protein structures, providing links between elements of secondary structure, and in many cases contributing to catalytic and binding sites. The conformations of short loops are now understood to depend primarily on their amino acid sequences. In contrast, the structural determinants of longer loops involve hydrogen-bonding and packing interactions within the loop and with other parts of the protein. By searching solved protein structures for regions similar in main chain conformation to the antigen-binding loops in immunoglobulins, we identified medium-sized loops of similar structure in unrelated proteins, and compared the determinants of their conformations. For loops that form compact substructures the major determinant of the conformation is the formation of hydrogen bonds to inward-pointing main chain atoms. For loops that have more extended conformations, the major determinant of their structure is the packing of a particular residue or residues against the rest of the protein. The following picture emerges: Medium-sized loops of similar conformation are stabilized by similar interactions. The groups that interact with the loop have very similar spatial dispositions with respect to the loop. However, the residues that provide these interactions may arise from dissimilar parts of the protein: The conformation of the loop requires certain interactions that the protein may provide in a variety of ways.     

Solid-phase synthesis of oligo-2-pyrimidinone-2'-deoxyribonucleotides and oligo-2-pyrimidinone-2'-deoxyriboside methylphosphonates.

A synthetic method was developed for the synthesis of oligodeoxyribonucleotides and oligodeoxyribonucleoside methylphosphonates comprised exclusively of the fluorescent 2-pyrimidinone base for the first time. The method utilized the solid-phase 2-cyanoethylphosphoramidite and methylphosphonamidite chemistry for internucleotide couplings and a baselabile oxalyl linkage to anchor the oligomers onto the CPG support. Cleavage of the oligomers from the support was effected by a short treatment of the support with 5% ammonium hydroxide in methanol at room temperature, without any degradation of the base-sensitive 2-pyrimidinone residues or the base-sensitive methylphosphonate backbone.     

Structural compromise of disallowed conformations in peptide and protein structures

Using a data set of 454 crystal structures of peptides and 80 crystal structures of non-homologous proteins solved at ultra high resolution of 1.2 A or better we have analyzed the occurrence of disallowed Ramachandran (phi, psi) angles. Out of 1492 and 13508 non-glycyl residues in peptides and proteins respectively 12 and 76 residues in the two datasets adopt clearly disallowed combinations of Ramachandran angles. These examples include a number of conformational points which are far away from any of the allowed regions in the Ramachandran map. According to the Ramachandran map a given (phi, psi) combination is considered disallowed when two non-bonded atoms in a system of two-linked peptide units with ideal geometry are prohibitively proximal in space. However, analysis of the disallowed conformations in peptide and protein structures reveals that none of the observations of disallowed conformations in the crystal structures correspond to a short contact between non-bonded atoms. A further analysis of deviations of bond lengths and angles, from the ideal peptide geometry, at the residue positions of disallowed conformations in the crystal structures suggest that individual bond lengths and angles are all within acceptable limits. Thus, it appears that the rare tolerance of disallowed conformations is possible by gentle and acceptable deviations in a number of bond lengths and angles, from ideal geometry, over a series of bonds resulting in a net gross effect of acceptable non-bonded inter-atomic distances.     

UV-induced cross-linking of proteins to plasmid pBR322 containing 8-azidoadenine 2'-deoxyribonucleotides

An efficient method of cross-linking DNA to protein is described. The method is based on the incorporation of photoactive 8-azidoadenine 2'-deoxyribonucleotides into DNA. We have found that 8-N3dATP is a substrate for E. coli DNA polymerase I and that 8-N3dATP can be incorporated into plasmid pBR322 by nick-translation. Subsequently we were able to cross-link a set of different proteins to 8-azido-2'-deoxyadenosine-containing pBR322 by UV irradiation (366nm). No DNA-protein photocross-linking was observed under the same conditions when the non-photoactive plasmid pBR322 was used.     

Charges of phosphate groups. A role in stabilization of 2'-deoxyribonucleotides. A DFT investigation

We have analyzed the relative stabilities and Gibbs tautomeric free energy for tautomeric transitions of neutral 2'-deoxyribonucleotides and its mono- and di-protonated forms. Geometry optimizations of these nucleic acid constituents have been performed at the DFT/B3LYP level using the standard 6-31G(d) basis set. The prediction of relative stabilities, Gibbs tautomeric free energy has been made at the B3LYP/6-311++G(d,p)//B3LYP/6-31G(d) level of theory. For each nucleoside four major conformers, i.e., north/anti, north/syn, south/anti, and south/syn have been taken into consideration. We have found the substantial effect of the uncompensated charge on the relative stability of 2'-deoxyribonucleotides. In particular, when the charge of 2'-deoxyribonucleotide anions is completely compensated by protons, the syn conformations have been found to be the global minima due to stabilization provided by intramolecular hydrogen bonds. However, the negative charge that appears due to the successive removal of the protons from the phosphate group destabilizes these syn conformations and stabilizes preferably the south/anti conformations (except of 2'-deoxyguanosine phosphate). Only 2'-deoxyribonucleotides, possessing south/anti and north/anti orientations, containing guanine and cytosine can contribute significantly to the rate of spontaneous point mutations due to the formation of biologically relevant amounts of 'rare' tautomers. However, we found strong influence of uncompensated negative charge for 2'-deoxyribonucleotides which possess syn conformations. Finally we have found that the proton transfer could result in the spontaneous change of 2'-deoxyribonucleotides conformations. We conclude that this phenomenon could be considered as a new way for the stabilization of 'rare' isomers for such DNA bases as cytosine and thymine.     

Structural flexibility in proteins: impact of the crystal environment

MOTIVATION: In the study of the structural flexibility of proteins, crystallographic Debye-Waller factors are the most important experimental information used in the calibration and validation of computational models, such as the very successful elastic network models (ENMs). However, these models are applied to single protein molecules, whereas the experiments are performed on crystals. Moreover, the energy scale in standard ENMs is undefined and must be obtained by fitting to the same data that the ENM is trying to predict, reducing the predictive power of the model. RESULTS: We develop an elastic network model for the whole protein crystal in order to study the influence of crystal packing and lattice vibrations on the thermal fluctuations of the atom positions. We use experimental values for the compressibility of the crystal to establish the energy scale of our model. We predict the elastic constants of the crystal and compare with experimental data. Our main findings are (1) crystal packing modifies the atomic fluctuations considerably and (2) thermal fluctuations are not the dominant contribution to crystallographic Debye-Waller factors. AVAILABILITY: The programs developed for this work are available as supplementary material at Bioinformatics Online.     

Structural and dynamic insights into α-synuclein dimer conformations

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2-micrometers DNA-like plasmids in the osmophilic haploid yeast Saccharomyces rouxii.

DNA plasmids were detected in two independent strains of Saccharomyces rouxii among 100 yeast strains other than Saccharomyces cerevisiae tested. The plasmids, pSR1 and pSR2, had almost the same mass (approximately 4 X 10(6) daltons) as 2-micrometers DNA of S. cerevisiae. pSR1 and pSR2 gave identical restriction maps with restriction endonucleases BamHI, EcoRI, HincII, HindIII, and XhoI, and both lacked restriction sites for PstI, SalI, and SmaI. These maps, however, differed significantly from that of S. cerevisiae 2-micrometers DNA. Restriction analysis also revealed two isomeric forms of each plasmid and suggested the presence of a pair of inverted repeat sequences in the molecules where intramolecular recombination took place. DNA-DNA hybridization between the pSR1 and pSR2 DNAs indicated significant homology between their base sequences, whereas no homology was detected between pSR1 and pJDB219, a chimeric plasmid constructed from a whole molecule of 2-micrometers DNA, plasmid pMB9, and a 1.2-kilobase DNA fragment of S. cerevisiae bearing the LEU2 gene. A chimeric plasmid constructed with pSR1 and YIp1, the larger EcoRI-SalI fragment of pBR322 ligated with a 6.1-kilobase DNA fragment of S. cerevisiae bearing the HIS3 gene, could replicate autonomously in an S. cerevisiae host and produced isomers, presumably by intramolecular recombination at the inverted repeats.     

Mechanical properties of DNA-like polymers

The molecular structure of the DNA double helix has been known for 60 years, but we remain surprisingly ignorant of the balance of forces that determine its mechanical properties. The DNA double helix is among the stiffest of all biopolymers, but neither theory nor experiment has provided a coherent understanding of the relative roles of attractive base stacking forces and repulsive electrostatic forces creating this stiffness. To gain insight, we have created a family of double-helical DNA-like polymers where one of the four normal bases is replaced with various cationic, anionic or neutral analogs. We apply DNA ligase-catalyzed cyclization kinetics experiments to measure the bending and twisting flexibilities of these polymers under low salt conditions. Interestingly, we show that these modifications alter DNA bending stiffness by only 20%, but have much stronger (5-fold) effects on twist flexibility. We suggest that rather than modifying DNA stiffness through a mechanism easily interpretable as electrostatic, the more dominant effect of neutral and charged base modifications is their ability to drive transitions to helical conformations different from canonical B-form DNA.     

Structural analysis of cytochromes P450 shows differences in flexibility of heme 2- and 4-vinyls

Structural analysis of the orientations of heme vinyl side chains was carried out using the published crystallographic data for different cytochromes P450. Torsional angles (tau, C(alpha)C(beta)-C(a)C(b)) show different distributions for the vinyls in positions 2 and 4. Whereas the orientation of 2-vinyls is rather restricted (tau between -120 degrees and -180 degrees ), the 4-vinyls have a much higher mobility over almost the entire conformational space. On the basis of the empirical correlation recently reported for peroxidases (M.P. Marzocchi, G. Smulevich, Relationship between heme vinyl conformation and the protein matrix in peroxidases, J. Raman Spectrosc. 34 (2003), 725-736), an attempt has been made to compare the observed vinyl orientations with the experimental frequencies of the vinyl stretching vibrational modes. The data for P450 proteins do not exactly match the peroxidase-derived function, although a qualitatively similar relationship is likely to exist. Differences between P450 forms suggest a variability in heme-region flexibility and in communication with the rest of enzyme.     

A and Z canonical conformations in d(CnGCGn) crystals characterized by microFTIR and microRaman spectroscopies

Two crystals d(C₂GCG₂) and d(C₅GCG₅) have been studied under microscope by Fourier transform ir spectroscopy and Raman spectroscopy. The x-ray diffraction study of the latter crystal had shown that the d(C₅GCG₅) sequence is the first DNA dodecamer known to adopt a canonical A conformation [N. Verdaguer, J. Aymami, D. Fernandez-Forner, I. Fita, M. Coll, T. Huynh-Dinh, J. Igolen, and J. A. Subirana (1991) Journal of Molecular Biology, Vol. 221, pp. 623–635]. Characteristic ir marker bands and Raman marker peaks of the A conformation have thus been obtained and are compared with previously proposed assignments correlated to fiber diffraction x-ray results obtained on polymers. The d(C₂GCG₂) sequence crystal had previously been studied in an intermediate form between B and Z [L. Urpi, J. P. Ridoux, J. Liquier, N. Verdagner, I. Fita, J. A. Subirana, F. Iglesias, T. Huynh-Dinh, J. Igolen, and E. Taillandier (1989) Nucleic Acids Research, Vol. 17, pp. 6669–6679]. In this paper we present results obtained from a crystal with this oligonucleotide in Z conformation. The effect of the crystallization conditions on the geometry of the obtained oligomer helix is discussed. The influence of the addition, to the central tetramer CGCG, of dC_n stretches (at the 5′ end) and dG_n stretches (at the 3′ end) of different lengths, on the conformational flexibility of the nucleic acid, is considered. © 1993 John Wiley & Sons, Inc.     

Backbone conformations and side chain flexibility of two somatostatin mimics investigated by molecular dynamics simulations

Molecular dynamics simulations with two designed somatostatin mimics, SOM230 and SMS 201‐995, were performed in explicit water for a total aggregated time of 208 ns. Analysis of the runs with SOM230 revealed the presence of two clusters of conformations. Strikingly, the two sampled conformers correspond to the two main X‐ray structures in the asymmetric unit of SMS 201‐995. Structural comparison between the residues of SOM230 and SMS 201‐995 provides an explanation for the high binding affinity of SOM230 to four of five somatostatin receptors. Similarly, cluster analysis of the simulations with SMS 201‐995 shows that the backbone of the peptide interconverts between its two main crystallographic conformers. The conformations of SMS 201‐995 sampled in the two clusters violated two different sets of NOE distance constraints in agreement with a previous NMR study. Differences in side chain fluctuations between SOM230 and SMS 201‐995 observed in the simulations may contribute to the relatively higher binding affinity of SOM230 to most somatostatin receptors. Proteins 2009. © 2008 Wiley‐Liss, Inc.