Factors governing 3(10)-helix vs alpha-helix formation in peptides: percentage of C(alpha)-tetrasubstituted alpha-amino acid residues and sequence dependence

As an additional step toward the dissection of the factors responsible for the onset of 3(10)-helix vs alpha-helix in peptides, in this paper we describe the results of a three-dimensional (3D) structural analysis by x-ray diffraction of the N(alpha)-acylated heptapeptide alkylamide mBrBz-L-Iva-L-(alphaMe)Val-L-Abu-L-(alphaMe)Val-L-(alphaMe)Phe-L-(alphaMe)Val-L-Iva-NHMe characterized by a single (L-Abu3) C(alpha)-trisubstituted and six C(alpha)-tetrasubstituted alpha-amino acids. We find that in the crystal state this peptide is folded in a mixed helical structure with short elements of 3(10)-helix at either terminus and a central region of alpha-helix. This finding, taken together with the published NMR and x-ray diffraction data on the all C(alpha)-methylated parent sequence and its L-Val2 analog (also the latter heptapeptide has a single C(alpha)-trisubstituted alpha-amino acid) strongly supports the view that one C(alpha)-trisubstituted alpha-amino acid inserted near the N-terminus of an N(alpha)-acylated heptapeptide alkylamide sequence may be enough to switch a regular 3(10)-helix into an essentially alpha-helical conformation. As a corollary of this work, the x-ray diffraction structure of the N(alpha)-protected, C-terminal tetrapeptide alkylamide Z-L-(alphaMe)Val-L-(alphaMe)Phe-L-(alphaMe)Val-L-Iva-NHMe, also reported here, is clearly indicative of the preference of this fully C(alpha)-methylated, short peptide for the 3(10)-helix. As the same terminally blocked sequence is mixed 3(10)/alpha-helical in the L-Abu3 heptapeptide amide but regular 3(10)-helical in the tetrapeptide amide and in the parent heptapeptide amide, these results point to an evident plasticity even of a fully C(alpha)-methylated short peptide.     

An analysis approach to identify specific functional sites in orthologous proteins using sequence and structural information: Application to neuroserpin reveals regions that differentially regulate inhibitory activity

The analysis of sequence conservation is commonly used to predict functionally important sites in proteins. We have developed an approach that first identifies highly conserved sites in a set of orthologous sequences using a weighted substitution‐matrix‐based conservation score and then filters these conserved sites based on the pattern of conservation present in a wider alignment of sequences from the same family and structural information to identify surface‐exposed sites. This allows us to detect specific functional sites in the target protein and exclude regions that are likely to be generally important for the structure or function of the wider protein family. We applied our method to two members of the serpin family of serine protease inhibitors. We first confirmed that our method successfully detected the known heparin binding site in antithrombin while excluding residues known to be generally important in the serpin family. We next applied our sequence analysis approach to neuroserpin and used our results to guide site‐directed polyalanine mutagenesis experiments. The majority of the mutant neuroserpin proteins were found to fold correctly and could still form inhibitory complexes with tissue plasminogen activator (tPA). Kinetic analysis of tPA inhibition, however, revealed altered inhibitory kinetics in several of the mutant proteins, with some mutants showing decreased association with tPA and others showing more rapid dissociation of the covalent complex. Altogether, these results confirm that our sequence analysis approach is a useful tool that can be used to guide mutagenesis experiments for the detection of specific functional sites in proteins. Proteins 2015; 83:135–152. © 2014 Wiley Periodicals, Inc.     

Facile transition between 3(10)- and alpha-helix: structures of 8-, 9-, and 10-residue peptides containing the -(Leu-Aib-Ala)2-Phe-Aib- fragment.

A structural transition from a 3(10)-helix to an alpha-helix has been characterized at high resolution for an octapeptide segment located in 3 different sequences. Three synthetic peptides, decapeptide (A) Boc-Aib-Trp-(Leu-Aib-Ala)2-Phe-Aib-OMe, nonapeptide (B) Boc-Trp-(Leu-Aib-Ala)2-Phe-Aib-OMe, and octapeptide (C) Boc-(Leu-Aib-Ala)2-Phe-Aib-OMe, are completely helical in their respective crystals. At 0.9 A resolution, R factors for A, B, and C are 8.3%, 5.4%, and 7.3%, respectively. The octapeptide and nonapeptide form ideal 3(10)-helices with average torsional angles phi(N-C alpha) and psi(C alpha-C') of -57 degrees, -26 degrees C and -60 degrees, -27 degrees for B. The 10-residue peptide (A) begins as a 3(10)-helix and abruptly changes to an alpha-helix at carbonyl O(3), which is the acceptor for both a 4-->1 hydrogen bond with N(6)H and a 5-->1 hydrogen with N(7)H, even though the last 8 residues have the same sequence in all 3 peptides. The average phi, psi angles in the decapeptide are -58 degrees, -28 degrees for residues 1-3 and -63 degrees, -41 degrees for residues 4-10. The packing of helices in the crystals does not provide any obvious reason for the transition in helix type. Fourier transform infrared studies in the solid state also provide evidence for a 3(10)- to alpha-helix transition with the amide I band appearing at 1,656-1,657 cm-1 in the 9- and 10-residue peptides, whereas in shorter sequences the band is observed at 1,667 cm-1.     

Computational study of the putative active form of protein Z (PZa): sequence design and structural modeling

Although protein Z (PZ) has a domain arrangement similar to the essential coagulation proteins FVII, FIX, FX, and protein C, its serine protease (SP)-like domain is incomplete and does not exhibit proteolytic activity. We have generated a trial sequence of putative activated protein Z (PZa) by identifying amino acid mutations in the SP-like domain that might reasonably resurrect the serine protease catalytic activity of PZ. The structure of the activated form was then modeled based on the proposed sequence using homology modeling and solvent-equilibrated molecular dynamics simulations. In silico docking of inhibitors of FVIIa and FXa to the putative active site of equilibrated PZa, along with structural comparison with its homologous proteins, suggest that the designed PZa can possibly act as a serine protease.     

A secondary structural model of the 28S rRNA expansion segments D2 and D3 for Chalcidoid wasps (Hymenoptera: Chalcidoidea)

We analyze the secondary structure of two expansion segments (D2, D3) of the 28S ribosomal (rRNA)-encoding gene region from 527 chalcidoid wasp taxa (Hymenoptera: Chalcidoidea) representing 18 of the 19 extant families. The sequences are compared in a multiple sequence alignment, with secondary structure inferred primarily from the evidence of compensatory base changes in conserved helices of the rRNA molecules. This covariation analysis yielded 36 helices that are composed of base pairs exhibiting positional covariation. Several additional regions are also involved in hydrogen bonding, and they form highly variable base-pairing patterns across the alignment. These are identified as regions of expansion and contraction or regions of slipped-strand compensation. Additionally, 31 single-stranded locales are characterized as regions of ambiguous alignment based on the difficulty in assigning positional homology in the presence of multiple adjacent indels. Based on comparative analysis of these sequences, the largest genetic study on any hymenopteran group to date, we report an annotated secondary structural model for the D2, D3 expansion segments that will prove useful in assigning positional nucleotide homology for phylogeny reconstruction in these and closely related apocritan taxa.     

Chromosomal organization of simple sequence repeats in the Pacific oyster (<Emphasis Type="Italic">Crassostrea gigas</Emphasis>): (GGAT)<Subscript>4</Subscript>, (GT)<Subscript>7</Subscript> and (TA)<Subscript>10</Subscript> chromosome patterns

Chromosome identification is essential in oyster genomic research. Fluorescence in situ hybridization (FISH) offers new opportunities for the identification of oyster chromosomes. It has been used to locate satellite DNAs, telomeres or ribosomal DNA sequences. However, regarding chromosome identification, no study has been conducted with simple sequence repeats (SSRs). FISH was used to probe the physical organization of three particular SSRs, (GGAT)(4), (GT)(7) and (TA)(10) onto metaphase chromosomes of the Pacific oyster, Crassostrea gigas. Hybridization signals were observed in all the SSR probes, but the distribution and intensity of signals varied according to the oligonucleotide repeat. The intercalary, centromeric and telomeric bands were observed along the chromosomes, and for each particular repeat every chromosome pair presented a similar pattern, allowing karyotypic analysis with all the SSRs tested. Our study is the first in mollusks to show the application of SSR in situ hybridization for chromosome identification and karyotyping. This technique can be a useful tool for oyster comparative studies and to understand genome organization in different oyster taxa.     

The complete mitochondrial genome sequence analysis of Tibetan argali (Ovis ammon hodgsoni): Implications of Tibetan argali and Gansu argali as the same subspecies

The genus Ovis (Bovidae, Artiodactyla) includes six species, i.e. Ovis ammon, Ovis aries, Ovis canadensis, Ovis dalli, Ovis nivicola and Ovis vignei. Based on morphology, geographical location, habitat, etc., the species O. ammon is divided into nine subspecies. The near threatened Tibetan argali is distributed across the Tibetan Plateau and its peripheral mountains, and believed to be one of the O. ammon subspecies (O. a. hodgsoni). However, considering its morphological features and distributions, a question has been proposed by some researchers about the subspecies status of Tibetan argali. In this study, we employed complete mitochondrial DNA (mtDNA) to explore the phylogenetic relationship and population genetic structure of Tibetan argali. The results revealed that the nucleotide composition, gene arrangement and codon usage pattern of the mitochondrial genome of Tibetan argali are similar to those of other caprines. Phylogenetic analyses showed that Tibetan argali was clustered with O. ammon. Interestingly, five Tibetan argali individuals and one of the three Gansu argali (O. a. dalailamae) individuals were clustered in the same branch, which is a sister group to other two Gansu argali individuals. Together with morphological characteristics, our results suggested that Tibetan argali and Gansu argali may belong to the same subspecies (O. a. hodgsoni) of O. ammon, rather than two different subspecies.