Comparative X-ray crystallographic evidence for a β-bend conformation as the active structure for peptide T in T4 receptor recognition

A sequence similarity has been found between two segments of endothiapepsin (acid proteinase, 2APE), bovine pancreatic ribonuclease A, and peptide T, a segment of the gp120 protein of human inmmune deficiency virus (HIV), which has been implicated in blocking viral attachment to the T4 receptor. The two similar sequences of the acid proteinase enzyme are Leu-Ile-Asp-Ser-Ser-Ala-Tyr-Thr (residues 169–176) and Tyr-Thr-Gly-Ser-Leu-Asn-Tyr-Thr (residues 175–182). Since the X-ray crystallographic structures of the acid proteinase and ribonuclease are known, it has been possible to determine whether the three-dimensional structures of the segments are similar. Portions of both of the segments of acid proteinase are directly superimposable on the structure of the RNase A 19–26 segment. The fact that the three similar sequences from two completely unrelated proteins give rise to almost identical structures raises the possibility that these segments may be involved in nucleating the folding of these proteins. In addition, this provides further support for the concept that the octapeptide sequence of peptide T of HIV, which is also similar in sequence to the 19–26 sequence of RNase A, is also structurally similar to these residues, which adopt a -bend conformation. Furthermore, comparison of similarities and differences in the structure of these similar sequences provides an explanation for alterations in the biological activity of various truncated or substituted derivatives of peptide T and additional confirmation of the structural requirements for peptide T in T4-receptor recognition.     

Structure, dynamics, and insertion of a chloroplast targeting peptide in mixed micelles

Nuclear-encoded, chloroplast-destined proteins are synthesized with transit sequences that contain all information to get them inside the organelle. Different proteins are imported via a general protein import machinery, but their transit sequences do not share amino acid homology. It has been suggested that interactions between transit sequence and chloroplast envelope membrane lipids give rise to recognizable, structural motifs. In this study a detailed investigation of the structural, dynamical, and topological features of an isolated transit peptide associated with mixed micelles is described. The structure of the preferredoxin transit peptide in these micelles was studied by circular dichroism (CD) and multidimensional NMR techniques. CD experiments indicated that the peptide, which is unstructured in aqueous solution, obtained helical structure in the presence of the micelles. By NMR it is shown that the micelles introduced ill-defined helical structures in the transit peptide. Heteronuclear relaxation experiments showed that the whole peptide backbone is very flexible. The least dynamic segments are two N- and C-terminal helical regions flanking an unstructured proline-rich amino acid stretch. Finally, the insertion of the peptide backbone in the hydrophobic interior of the micelle was investigated by use of hydrophobic spin-labels. The combined data result in a model of the transit peptide structure, backbone dynamics, and insertion upon its interaction with mixed micelles.     

Methionine ligation strategy in the biomimetic synthesis of parathyroid hormones

In biological systems, both proteolysis and aminolysis of amide bonds produce activated intermediates through acyl transfer reactions either inter- or intramolecularly. Protein splicing is an illustrative example that proceeds through a series of catalyzed acyl transfer reactions and culminates at an O- or S-acyl intermediate. This intermediate leads to an uncatalyzed acyl migration to form an amide bond in the spliced product. A ligation method mimicking the uncatalyzed final steps in protein splicing has been developed utilizing the acyl transfer amide-bond feature for the blockwise coupling of unprotected, free peptide segments at methionine (Met). The latent thiol moiety of Met can be exploited using homocysteine at the α-amino terminal position of a free peptide for transthioesterification with another free peptide containing an α-thioester to give an S-acyl intermediate. A subsequent, proximity-driven S- to N-acyl migration of this acyl intermediate spontaneously rearranges to form a homocysteinyl amide bond. S-methylation with excess p-nitrobenezensulfonate yields Met at the ligation site. The methionine ligation is selective and orthogonal, and is usually completed within 4 h when performed at slightly basic pH and under strongly reductive conditions. No side reactions due to acylation were observed with any other α-amines of both peptide segments as seen in the synthesis of parathyroid hormone peptides. Furthermore, cyclic peptide can also be obtained through the same strategy by placing both homocysteine at the amino terminus and the thioester at the carboxyl terminus in an unprotected peptide precursor. These biomimetic ligation strategies hold promise for engineering novel peptides and proteins. © 1998 John Wiley & Sons, Inc. Biopoly 46: 319–327, 1998     

Simple sequence is abundant in eukaryotic proteins.

All proteins of Saccharomyces cerevisiae have been compared to determine how frequently segments from one protein are present in other proteins. Proteins that are recently evolutionarily related were excluded. The most frequently present protein segments are long, tandem repetitions of a single amino acid. For some of these segments, up to 14% of all proteins in the genome were found to have similar peptides within them. These peptide segments may not be functional protein domains. Although they are the most common shared feature of yeast proteins, their ubiquity and simplicity argue that their probable function may be to simply serve as spacers between other protein motifs.     

Tandem Peptide Ligation for Synthetic and Natural Biologicals

We describe the concept and methods of peptide ligation and tandem peptide ligation for preparing synthetic and natural biologicals. Peptide ligation is a segment coupling method for free peptides or proteins through an amide bond without the use of a coupling reagent or a protecting group scheme. Because unprotected peptides or proteins prepared from either a chemical or biochemical source are being used as building blocks, the ligation removes the size limitation for peptide and protein synthesis. A key feature of the peptide ligation is that the coupling reaction is orthogonal, i.e. it is specific to a particular alpha-amino terminus (NT). This NT-amino acid-specific feature permits the development of a tandem peptide ligation method employing three unprotected peptide segments containing different NT-amino acids to form consecutively two amide bonds, an Xaa-SPro (thiaproline) and then an Xaa-Cys. This strategy was tested in peptides ranging from 28 to 70 amino acid residues, including analogues of somatostatins and two CC-chemokines MIP-1alpha and MIP-1beta. The thiaproline replacements in these peptides and proteins did not result in altered biological activity. By eliminating the protecting group scheme and coupling reagents, tandem ligation of multiple free peptide segments in aqueous solutions enhances the scope of protein synthesis and may provide a useful approach for preparing protein biologicals and synthetic vaccines.     

Peptide mimics of the M13 coat protein transmembrane segment. Retention of helix-helix interaction motifs

Sequence-specific noncovalent helix-helix interactions between transmembrane (TM) segments in proteins are investigated by incorporating selected TM sequences into synthetic peptides using the construct CKKK-TM-KKK. The peptides are of suitable hydrophobicity for spontaneous membrane insertion, whereas formation of an N-terminal S-S bond can bring pairs of TM helices into proximity and promote their parallel orientation. Using the propensity of the protein to undergo thermally induced alpha-helix --> beta-sheet transitions as a parameter for helix stability, we compared the wild type and mutant (V29A and V31A) bacteriophage M13 coat proteins with their corresponding TM peptide constructs (M13 residues 24-42). Our results demonstrated that the relevant helix-helix tertiary contacts found in the intact proteins persist in the peptide mimics. Molecular dynamics simulations support the tight "two in-two out" dimerization motif for V31A consistent with mutagenesis data. The overall results reinforce the notion of TM segments as autonomous folding domains and suggest that the generic peptide construct provides a viable reductionist system for membrane protein structural and computational analysis.     

Using a neural network to identify potential HLA-DR1 binding sites within proteins

The presentation by antigen-presenting cells of immunodominant peptide segments in association with major histocompatibility complex (MHC) encoded proteins is fundamental to the efficacy of a specific immune response. One approach used to identify immunodominant segments within proteins has involved the development of predictive algorithms which utilize amino acid sequence data to identify structural characteristics or motifs associated with in vivo antigenicity. The parallel-computing technique termed ‘neural networking’ has recently been shown to be remarkably efficient at addressing the problem of pattern recognition and can be applied to predict protein secondary structure attributes directly from amino acid sequence data. In order to examine the potential of a neural network to generalize peptide structural feature related to binding within class II MHC-encoded proteins, we have trained a neural network to determine whether or not any given amino acid of a protein is part of a peptide segment capable of binding to HLA-DR1. We report that a neural network trained on a data base consisting of peptide segments known to bind to HLA-DR1 is able to generalize features relating to HLA-DR1-binding capacity (r = 0.17 and p = 0.0001).     

Folding of the nascent peptide chain into a biologically active protein

The refolding of denatured proteins with complete sequences may not be fast enough to account for the in vivo folding of growing peptide chains during biosynthesis. As some peptide fragments have secondary structures not unlike those of the corresponding segments in the intact molecules and native disulfide bonds of some proteins can form cotranslationally, it is suggested that the folding of the nascent chain begins early during synthesis. However, further adjustments may be necessary during chain elongation and after posttranslational modifications of the completed peptide chain to generate the native conformation of a biologically active protein.     

Stability of loops in the structure of lactose permease

Structural analysis of peptide fragments has provided useful information on the secondary structure of integral membrane proteins built from a helical bundle (up to seven transmembrane segments). Comparison of those results to recent X-ray crystallographic results showed agreement between the structures of the fragments and the structures of the intact proteins. Lactose permease of Escherichia coli (lac Y) offers an opportunity to test that hypothesis on a substantially larger integral membrane protein. Lac Y contains a bundle of 12 transmembrane segments connected by 11 loops. Eleven segments, each corresponding to one of the loops in this protein, were studied. Five of these segments form defined structures in solution as determined by multidimensional nuclear magnetic resonance. Four peptides form turns, and one peptide reveals the end of one of the transmembrane helices. These results suggest that some loops in helical bundles are stabilized by short-range interactions, particularly in smaller bundles, and such intrinsically stable loops may contribute to protein stability and influence the pathway of folding. Greater conformational flexibility may be found in large integral membrane proteins.     

Nucleotide sequence of pig plasma gelsolin. Comparison of protein sequence with human gelsolin and other actin-severing proteins shows strong homologies and evidence for large internal repeats

Pig plasma gelsolin (Mr = 81595; 739 residues) contains 704 identical residues out of a maximum 730 when compared to the cytoplasmic form of human gelsolin. The cDNA sequence also codes for a peptide of 33 residues N-terminal to the nine-residue plasma extension sequence previously reported: these 33 residues are highly homologous to the human signal peptide and plasma extension. Comparison of the gelsolin sequences with chicken brush border villin, severin from Dictyostelium discoideum and fragmin from Physarum polycephalum shows a strong evolutionary relationship between all these proteins. There are six large repeating segments in gelsolin and villin, and three similar segments in severin and fragmin. Although these multiple repeats cannot be related to any known function of these actin-severing proteins, this superfamily of proteins appears to have evolved from an ancestral sequence of 120 to 130 amino acid residues.     

Identification of structured peptide segments in folding proteins.

Prediction of the structures of long polypeptides and small proteins has been carried out using conformational energy calculations. These calculations can be applied to large proteins if structured regions of their sequences can be identified. Three different approaches to identifying such sequences are presented. First, sequences of five or more contiguous hydrophobic residues tend to nucleate alpha-helices. Second, peptide sequences from parent proteins that have the same biological activities as the parent proteins are highly structured. Third, structured synthetic peptide segments from proteins inhibit the folding of the parent proteins by competing with the corresponding segment of the protein chain for associating with complementary regions. Examples of each of these approaches are presented.     

A new approach to secondary structure evaluation: Secondary structure prediction of porcine adenylate kinase and yeast guanylate kinase by CD spectroscopy of overlapping synthetic peptide segments

A new approach for evaluating the secondary structure of proteins by CD spectroscopy of overlapping peptide segments is applied to porcine adenylate kinase (AK1) and yeast guanylate kinase (GK3). One hundred seventy-six peptide segments of a length of 15 residues, overlapping by 13 residues and covering the complete sequences of AK1 and GK3, were synthesized in order to evaluate their secondary structure composition by CD spectroscopy. The peptides were prepared by solid phase multiple peptide synthesis method using the 9-fluorenylmethoxycarbonyl/tert-butyl strategy. The individual peptide secondary structures were studied with CD spectroscopy in a mixture of 30% trifluoroethanol in phosphate buffer (pH 7) and subsequently compared with x-ray data of AK1 and GK3. Peptide segments that cover α-helical regions of the AK1 or GK3 sequence mainly showed CD spectra with increasing and decreasing Cotton effects that were typical for appearing and disappearing α-helical structures. For segments with dominating β-sheet conformation, however, the application of this method is limited due to the stability and clustering of β-sheet segments in solution and due to the difficult interpretation of random-coiled superimposed β-sheet CD signals. Nevertheless, the results of this method especially for α-helical segments are very impressive. All α-helical and 71% of the β-sheet containing regions of the AK1 and GK3 could be identified. Moreover, it was shown that CD spectra of consecutive peptide content reveal the appearance and disappearance of α-helical secondary structure elements and help localizing them on the sequence string. © 1997 John Wiley & Sons, Inc. Biopoly 41: 213–231, 1997     

Amino acid sequence pattern in the regulatory peptides.

The essential properties of the primary structure of regulatory peptides, i.e. amino acid residues and their combinations, which are characteristic of the whole population of regulatory peptides, have been revealed using statistical methodology. These properties are as follows: increased content of certain residues (Gly, Pro, Phe, Arg, Tyr, Met and Trp) as well as an increased rate of occurrence of certain pairs of residue as compared with proteins, a random sequence of residues and "nonregulatory" peptides. By representing regulatory peptides as a sequence of hydrophobic (2 types) and hydrophilic (3 types) segments, the pattern for alternation of these segments in regulatory peptides has been determined. The segments were classified into 5 types according to the peculiarities of mutual localization of hydrophobic and hydrophilic residues within the primary structure of regulatory peptides. As compared with proteins, "nonregulatory" peptides and a random sequence of segments, regulatory peptides were characterized by an increased frequency of 4 particular pairs of segments among 12 theoretically possible pairs. These 4 pairs are fragments of the periodic segment sequence with periods of 4 segments. The revealed pattern indicates that there exists a general principle of the regulatory peptide primary structure organization and possibly a common type of the regulatory peptides flexible peptide chain folding at the ligand-receptor complex formation.     

Strategies for dealing with conformational sampling in structural calculations of flexible or kinked transmembrane peptides.

Peptides corresponding to transmembrane (TM) segments from membrane proteins provide a potential route for the determination of membrane protein structure. We have determined that 2 functionally critical TM segments from the mammalian Na+/H+ exchanger display well converged structure in regions separated by break points. The flexibility of these break points results in conformational sampling in solution. A brief review of available NMR structures of helical membrane proteins demonstrates that there are a number of published structures showing similar properties. Such flexibility is likely indicative of kinks in the full-length protein. This minireview focuses on methods and protocols for NMR structure calculation and analysis of peptide structures under conditions of conformational sampling. The methods outlined allow the identification and analysis of structured peptides containing break points owing to conformational sampling and the differentiation between oligomerization and ensemble-averaged observation of multiple peptide conformations.     

枯草芽孢杆菌同义密码子使用偏性对蛋白质折叠速率的影响

目前,有关同义密码子使用偏性对蛋白质折叠的影响研究中,样本蛋白均来源于不同的物种。考虑到同义密码子使用偏性的物种差异性,选取枯草杆菌的核蛋白为研究对象。首先,将每条核蛋白按二级结构截取为α螺旋片段、β折叠片段和无规卷曲（α-β混合）片段,并计算其蛋白质折叠速率。然后,整理每个片段相应的核酸序列信息,计算其同义密码子使用度。在此基础上,分析枯草芽孢杆菌核蛋白的同义密码子使用偏性与蛋白质折叠速率的相关性。发现对于不同二级结构的肽链片段,都有部分密码子的使用偏性与其对应的肽链折叠速率显著相关。进一步分析发现,与肽链片段折叠速率显著相关的密码子绝大部分为枯草杆菌全序列或核蛋白序列的每一组同义密码子中使用度最高的密码子。结果表明,在蛋白质的折叠过程中,枯草芽孢杆菌的同义密码子使用偏性起着重要作用。     

Polypeptide synthesis by the thioester method

A novel method for polypeptide synthesis, in which partially protected peptide thioesters are used as building blocks, has been developed. Partially protected peptide thioesters are easily prepared by solid-phase methodology. The thioester moiety is converted to an active ester in the presence of a silver compound such as AgNO(3) or AgCl and an active ester component such as 1-hydroxybenzotriazole or 3,4-dihydro-3-hydro-4-oxo-1,2, 3-benzotriazine. Segment condensation can be accomplished using partially protected peptide segments. The consecutive condensation of the partially protected peptide segments is realized by the selective removal of the 9-flourenylmethoxycarbonyl group, for terminal amino protection, after segment condensation has been achieved. In this method, large peptide segments can easily be used. Thus, the products obtained by the thioester method can be separated from by-products by reverse phase high performance liquid chromatography, even when no purification process was performed during the prior segment condensation procedures. This indicates that proteins that have no specific features such as enzymatic or biological activities can be obtained after isolation, solely based on their chromatographic profiles. Thus, the thioester method will provide a new basis for protein studies including phosphorylated and glycosylated polypeptides.     

Chemical synthesis and semisynthesis of membrane proteins

Total chemical synthesis and semisynthesis of proteins have become widely used tools to alter and control the chemical structure of soluble proteins, Thus, offering unique possibilities to understand protein function in vitro and in vivo. However, these approaches rely on our ability to produce and chemoselectively link peptide segments with each other or with recombinantly produced protein segments. Access to integral membrane and membrane-associated proteins via these approaches has been hampered by the fact that integral membrane peptides or lipid-modified peptides are difficult to obtain mostly due to incomplete amino acid coupling reactions and their poor handling properties. This article will highlight the advances in the total chemical synthesis and semisynthesis of small viral as well as bacterial ion channels. Recent synthesis approaches for membrane-associated proteins will be discussed as well.     

Co-translational folding

Nascent proteins appear to fold co-translationally. The ribosome itself may function as a chaperone, providing a sheltered environment in which the nascent peptide is protected from aggregation and degradation, and in which folding into the tertiary structure is facilitated by interactions both with ribosomal proteins and with specific segments of the ribosomal RNA.     

Asparagine-mediated self-association of a model transmembrane helix

In membrane proteins, the extent to which polarity, hydrogen bonding, and van der Waals packing interactions of the buried, internal residues direct protein folding and association of transmembrane segments is poorly understood. The energetics associated with these various interactions should differ substantially between membrane versus water-soluble proteins. To help evaluate these energetics, we have altered a water-soluble, two-stranded coiled-coil peptide to render its sequence soluble in membranes. The membrane-soluble peptide associates in a monomer-dimer-trimer equilibrium, in which the trimer predominates at the highest peptide/detergent ratios. The oligomers are stabilized by a buried Asn side chain. Mutation of this Asn to Val essentially eliminates oligomerization of the membrane-soluble peptide. Thus, within a membrane-like environment, interactions involving a polar Asn side chain provide a strong thermodynamic driving force for membrane helix association.     

Evaluation of a pepscan approach to identify epitopes recognised by anti-hTSH monoclonal antibodies

In this study, several methodological aspects of the pepscan strategy have been investigated with the objective to delineate the amino acid sequences of peptide segments that form the epitopes of thyrotropin beta-subunit (TSHbeta) recognised by monoclonal antibodies. Hitherto, the pepscan strategy has found application as an effective method to identify linear sequence regions that constitute contiguous epitopes within the primary structure of some proteins. However, with heterodimeric glycoprotein hormones and their subunits such as TSHbeta, as well as for many other globular proteins, the majority of the epitopes recognised by anti-protein antibodies will be derived from discontinuous segments that collectively form the epitope. In these cases the pepscan technique will only be able to identify individual segments of the overall discontinuous epitope site as linear peptides, some of which may interact with relatively low binding affinity. Consequently, additional attention must thus be given to the optimisation of the specific binding and detection conditions. Knowledge of the structures of these peptide segments can, however, provide a valuable basis to develop peptide structures that more closely mimic the topographical features of the epitope in the mature, folded protein. In an attempt to identify functional segments involved in the epitopes recognised by the anti-hTSH monoclonal antibodies, mAb279 and mAb299, the impact of various experimental conditions on the efficacy of the pepscan strategy has been investigated. The strategy involved the synthesis of a series of overlapping pin-bound octapeptides with amino acid sequences derived from the TSH beta-subunit. The ability of these pin-bound octapeptides to bind to either mAb279 or mAb299 in ELISA-based assay was then determined under conditions involving different concentrations of the primary and/or secondary antibodies, and changes in buffer composition, incubation times and washing procedures. Theresults of this study illustrate some of the constraints and limitations of the pepscan technique when used to delineate discontinuous epitopes of globular proteins, as well as providing insight into potential avenues to optimise and refine this method.