Amino Acid Residues In Peptides Research Articles

Crystal structure of vesicular stomatitis virus matrix protein.

The vesicular stomatitis virus (VSV) matrix protein (M) interacts with cellular membranes, self-associates and plays a major role in virus assembly and budding. We present the crystallographic structure, determined at 1.96 A resolution, of a soluble thermolysin resistant core of VSV M. The fold is a new fold shared by the other vesiculovirus matrix proteins. The structure accounts for the loss of stability of M temperature-sensitive mutants deficient in budding, and reveals a flexible loop protruding from the globular core that is important for self-assembly. Membrane floatation shows that, together with the M lysine-rich N-terminal peptide, a second domain of the protein is involved in membrane binding. Indeed, the structure reveals a hydrophobic surface located close to the hydrophobic loop and surrounded by conserved basic residues that may constitute this domain. Lastly, comparison of the negative-stranded virus matrix proteins with retrovirus Gag proteins suggests that the flexible link between their major membrane binding domain and the rest of the structure is a common feature shared by these proteins involved in budding and virus assembly.

Inhibitory effect of ghrelin on insulin and pancreatic somatostatin secretion

Ghrelin is a 28 amino acid residue peptide identified in both human and rat stomach and which acts as an endogenous ligand for the GH secretagogue receptor (GHS-R) and stimulates GH release. GHS-Rs are expressed in a number of tissues, including the pancreas, and ghrelin-like immunoreactivity is present in peripheral plasma, where its levels increase during fasting and decrease after food intake. The relationship between nutritional status and circulating ghrelin concentrations prompted us to investigate the effect of this peptide on pancreatic hormone secretion. The study was performed in the isolated rat pancreas perfused in situ. Insulin, glucagon and somatostatin were measured by radioimmunoassay. Addition of 10 nM ghrelin to the perfusate significantly reduced the insulin response to the secretagogues glucose, arginine and carbachol, which act on the B-cell via different mechanisms, as well as the somatostatin response to arginine. Ghrelin was without effect on the glucagon output induced by this amino acid. At a lower concentration (2 nM) ghrelin was also found to inhibit glucose-induced insulin release. These findings support the proposal that the inhibitory effect of ghrelin on insulin release constitutes a tonic regulation of the B-cell, contributing to restrain its secretory activity in the state of food deprivation. On the other hand, the inhibition of pancreatic somatostatin release by ghrelin suggests a blocking effect of this hormone on the widely distributed D-cell population.

Conformational dynamics of peptide T molecule

Using a method of the theoretical conformational analysis, a conformational dynamics of the side chains of the amino acid residues of peptide T, a competitor of the human immuno-deficiency virus in the binding to human T cells, was investigated. For this purpose, the conformational maps of the potential surfaces were constructed over the angles of the side chains for the preferable conformations of peptide T molecule. Permissible deviations of these angles from the optimal values were determined. It has been found that the angles of the side chains of the amino acid residues involved in physiologically active fragment Thr4-Thr8 are more rigid than in the other segment of the molecule. This fact confirms the existence of such a regular structure as β-turn revealed previously in studies of the spatial structure of the peptide T molecule.

Regio- and stereoselective α-alkylation of N-terminal amino acid residue of peptides using a pyridoxal model compound with a chiral ansa-structure

Regio- and stereoselective α-alkylation of N-terminal amino acid residue of peptides was achieved by Li +-mediated alkylation of aldimines prepared from the peptides and a pyridoxal model compound having a chiral ansa-structure and an ethoxyethoxy group at C-3. The stereochemistry and stereoselectivity of the reaction were found to be influenced predominantly by the chirality of the model compound and Li +, but little by the stereochemistry of the original peptides.

CHEMICAL CROSS-LINKING OF PEPTIDES DERIVED FROM RECA WITH SINGLE-STRANDED OLIGONUCLEOTIDES CONTAINING 5-FORMYL-2′-DEOXYURIDINE

We report the first example of chemical cross-linking of 5-formyl-2′-deoxy-uridine containing oligonucleotides with oligopeptides through a Schiff base formation. Twenty amino acid residue peptides investigated here were derived from the DNA binding site of RecA protein. We have demonstrated that the lysine residue placed at the 6th or 8th position from the N-terminus of the peptide directly contacts with DNA.

A peptide derived from the C‐terminal part of a plant cysteine protease folds into a stack of two β‐hairpins, a scaffold present in the emerging family of granulin‐like growth factors

A 35 amino acid residue peptide corresponding to the N-terminal subdomain of the granulin-like repeat from rice oryzain beta was synthesized and regioselectively oxidized to produce a species with a [1-3, 2-4] disulfide-pairing pattern. The resulting peptide was studied in solution using NMR and was shown to adopt the tertiary topology of a stack of two beta-hairpins found in the emerging family of granulin-like growth factors. Because of the longer second beta-hairpin, the overall conformation of the peptide is somewhat more flexible than that of its well-structured carp granulin-1 analog. Except for the cysteine alignment, there is very little sequence homology between granulin-like growth factors from the animal kingdom and the granulin-like repeats at the C-termini of plant cysteine proteases. Therefore, the stack of two beta-hairpins may be a conserved three-dimensional organization of the granulin-like repeats from evolutionary distant sources with a significant role in specific protein-protein interactions.

Differential Sensitivity to Mutations in a Single Peptide by Two TCRs Having Identical β-Chains and Closely Related α-Chains

The TCR on CD4 T cells binds to and recognizes MHC class II:antigenic peptide complexes through molecular contacts with the peptide amino acid residues that face up and out of the peptide-binding groove. This interaction primarily involves the complementarity-determining regions (CDR) of the TCR alpha- and ss-chains contacting up to five residues of the peptide. We have used two TCRs that recognize the same antigenic peptide and have identical Vss8.2 chains, but differ in all three CDR of their related Valpha2 chains, to examine the fine specificity of the TCR:peptide contacts that lead to activation. By generating a peptide library containing all 20 aa residues in the five potential TCR contact sites, we were able to demonstrate that the two similar TCRs responded differentially when agonist, nonagonist, and antagonist peptide functions were examined. Dual substituted peptides containing an agonist residue at the N terminus, which interacts with CDR2alpha, and an antagonist residue at the C terminus, which interacts with the CDR3ss, were used to show that the nature of the overall signal through the TCR is determined by a combination of the type of signal received through both the TCR alpha- and ss-chains.

Interaction of peptides derived from RecA with single-stranded oligonucleotides containing 5-formyl-2'-deoxyuridine.

We report the first example of chemical cross-linking of 5-formyl-2'-deoxyuridine containing oligonucleotides with oligopeptides through a Schiff base formation. Twenty amino acid residue peptides investigated here were derived from the DNA binding site of RecA protein. We have demonstrated that the lysine residue placed at the 6th or 8th position from the N-terminus of the peptide directly contacts with DNA.

Selection of peptidic mimics of digoxin from phage-displayed peptide libraries by anti-digoxin antibodies

Since the initial report of the development of methodology to generate high-affinity digitalis-specific (digoxin) antibodies, these antibodies have proven extremely useful tools to monitor digoxin levels in digitalized patients and, as Fab fragments, to reverse toxic digoxin effects in life-threatening digoxin overdoses. These antibodies (both digoxin-specific and ouabain-specific) have been used extensively by investigators for the identification and characterization of putative endogenous digitalis-like factors. In this study, we used two well-characterized mouse anti-digoxin monoclonal antibodies (mAbs), designated 26–10 and 45–20, as binding templates with which to select short bacteriophage-displayed (pIII protein inserted) peptides that are capable of binding to these mAbs and mimicking the conformational structure of digoxin. Selective enrichment from two phage-displayed random peptide libraries enabled us to isolate and identify distinct 15 and 26 amino acid residue peptide inserts that bind with high avidity and idiotypic specificity to the selecting mAbs. Among these displayed inserts a subset was identified whose mAb binding is inhibited by digoxin and whose corresponding synthetic peptides inhibit phage binding. They, therefore, appear to bind at the mAbs digoxin-binding sites. These data provide the first clear evidence that short polypeptides can serve as surrogates for the low molecular mass hapten digoxin.

The amide proton NMR chemical shift and hydrogen-bonded structure of peptides and polypeptides in the solid state as studied by high-frequency solid-state [formula omitted] NMR

High-resolution 1 H NMR spectra of glycine (Gly)-containing peptides and polypeptides in the solid state were measured at 800 MHz and at high-speed magic-angle-spinning (MAS) of 30 kHz to elucidate the relationship between the hydrogen-bond length and 1 H NMR chemical shift to add to our previous experimental and theoretical findings that there is a relationship between the hydrogen-bond length and 13 C , 15 N and 17 O chemical shifts of various kinds of amino acid residues of peptides and polypeptides in the solid state. From these experimental results, it is found that the 1 H chemical shifts of Gly amide protons of Gly-containing peptides and polypeptides, for which the hydrogen-bond length between the nitrogen and oxygen atoms ( R N…O) have already been determined by X-ray diffraction, move downfield with a decrease in R N…O. Theoretical calculations qualitatively explain these experimental results.

Hypertension in black patients: an emerging role of the endothelin system in salt-sensitive hypertension.

The prevalence of essential hypertension in blacks is much higher than that in whites. In addition, the pathogenesis of hypertension appears to be different in black patients. For example, black patients present with a salt-sensitive hypertension characterized by low renin levels. Racial differences in renal physiology and socioeconomic factors have been suggested as possible causes of this difference, but reasons for this difference remain unclear. Endothelial cells are important in the regulation of vascular tonus and homeostasis, in part through the secretion of vasoactive substances. One of these factors, endothelin-1 (ET-1), is a 21 amino acid residue peptide with potent vasopressor actions. In addition to its contractile effects, it has been shown to stimulate mitogenesis in a number of cell types. Moreover, ET-1 displays modulatory effects on the endocrine system, including stimulation of angiotensin II and aldosterone production and inhibition of antidiuretic hormone in the kidney. Recent data from several laboratories indicate that ET-1 is overexpressed in the vasculature in several salt-sensitive models of experimental hypertension. Moreover, circulating plasma ET-1 levels are significantly increased in black hypertensives compared with white hypertensives. Thus, the ET system might be particularly important in the development or maintenance of hypertension in this population.

Two N-terminally truncated forms of C-type natriuretic peptide from habu snake venom

Two N-terminally truncated forms of the C-type natriuretic peptide (CNP) were isolated from the venom of habu snake, Trimeresurus flavoviridis, and their structures were determined by EMI-MS spectrometry and amino acid sequencing. Tf-CNP(6–22), the shorter peptide retaining the 17-membered ring structure formed by an intra-molecular disulfide bridge, has a vasorelaxant activity in rat aortic strips and a diuretic potency in anesthetized rats. Tf-CNP(3–22), the other 20 amino acid residues peptide, also comprised the 17- membered ring with a short N-terminal extention of 3 amino acid residues. Tf-CNP(6–22), the ring, is the shortest naturally occurring CNP peptide identified so far, and as potent as Tf-CNP(1–22), the supposedly intact CNP of 22 amino acid residues.

Solution structure of hanatoxin1, a gating modifier of voltage-dependent K + channels: common surface features of gating modifier toxins

The three-dimensional structure of hanatoxin1 (HaTx1) was determined by using NMR spectroscopy. HaTx1 is a 35 amino acid residue peptide toxin that inhibits the drk1 voltage-gated K + channel not by blocking the pore, but by altering the energetics of gating. Both the amino acid sequence of HaTx1 and its unique mechanism of action distinguish this toxin from the previously described K + channel inhibitors. Unlike most other K + channel-blocking toxins, HaTx1 adopts an “inhibitor cystine knot” motif and is composed of two β-strands, strand I for residues 19–21 and strand II for residues 28–30, connected by four chain reversals. A comparison of the surface features of HaTx1 with those of other gating modifier toxins of voltage-gated Ca 2+ and Na + channels suggests that the combination of a hydrophobic patch and surrounding charged residues is principally responsible for the binding of gating modifier toxins to voltage-gated ion channels.

Identification of unusual (modified and non-encoded) amino acid residues in peptides by combinations of high-performance liquid chromatography and high-performance capillary electrophoresis with matrix-assisted laser desorption ionization time-of-flight mass spectrometry

The techniques for micro-level analysis of some widespread unusual amino acids (phosphorylated and hydroxylated ones) as well as of some genetically non-encoded amino acids were developed for their subsequent identification in the peptide and protein amino acid sequence by narrow-bore column high-performance liquid chromatography (∼10 pmol of the sample), high-performance capillary electrophoresis (∼1–10 pmol), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (∼1–10 pmol), and automatic protein gas phase sequencing (∼1–50 pmol).

A search within the IL-1 type I receptor reveals a peptide with hydropathic complementarity to the IL-1β trigger loop which binds to IL-1 and inhibits in vitro responses

In previous research, we were able to demonstrate that a seven amino acid residue peptide (VITFFSL), designed as an antisense peptide of the β-bulge trigger loop region of interleukin 1β (IL-1β) (QGEESND; residues 48–54 [mature protein sequence]), was able to interact with IL-1 specifically and inhibit the response to IL-1 in an in vitro bioassay. The evidence was consistent with a specific interaction ocurring between antisense peptide and the trigger loop region. On the basis that antisense peptides are able to interact with their corresponding sense peptide sequences as a result of their mutually complementary hydropathic profiles (Fassina G., Verdoliva, A., Cassani, G., Melli, M., 1994. Binding of type I IL-1 receptor fragment 151–162 to IL-1. Growth Factors 10, 99–106; Maier, C.C., Moseley, H.N.B., Zhou, S., Whitaker, J.N., Blalock, J.E., 1994. Indentification of interactive determinants on idiotypic-anti-idiotypic antibodies through comparison of their hydropathic profiles. Immunomethods 5, 107–113), we devised a computer program (FINDH) to search the amino acid residue sequence of interleukin-1 type 1 receptor (IL-1 R1) for peptide motifs possessing hydropathic complementarity to the trigger loop sequence. The most complementary “best-fit peptide” motif (LITVLNI) was located in the third extracellular domain of IL-1 R1. A best-fit peptide corresponding to this motif was synthesised and found to bind to IL-1β as well as inhibit the response to IL-1 in two independent in vitro bioassays (monitoring IL-1 dependent serum amyloid A synthesis and IL-1 dependent alkaline phosphatase activity, respectively). A second peptide motif (VIEFITL) was identified and the corresponding peptide synthesised along with a reordered version (LTILINV) of the best fit peptide. Both failed to bind measurably with IL-1β or inhibit the response to IL-1 in the two bioassays. This best fit peptide behaved very similarly, in terms of IL-1 binding and inhibition behaviour, to the original trigger loop antisense peptide. Reference to the recently released X-ray crystal structure of IL-1β and the IL1-R1 extracellular domain shows that the best fit peptide motif in IL-1 R1 is not apparantly interacting with the IL-1 trigger loop, although both are close in space. The intriguing possibility exists that the best fit peptide motif could represent an alternative site for IL-1β receptor interaction which has not thus far been identified.

MEARA sequence repeat of human CstF-64 polyadenylation factor is helical in solution. A spectroscopic and calorimetric study.

The primary structure of the human CstF-64 polyadenylation factor contains 12 nearly identical repeats of a consensus motif of five amino acid residues with the sequence MEAR(A/G). No known function has yet been ascribed to this motif; however, according to secondary structure prediction algorithms, it should form a helical structure in solution. To validate this theoretical prediction, we synthesized a 31 amino acid residue peptide (MEARA(6)) containing six repeats of the MEARA sequence and characterized its structure and stability by circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC). No effects of concentration on the CD or DSC properties of MEARA(6) were observed, indicating that the peptide is monomeric in solution at concentrations up to 2 mM. The far UV-CD spectra of MEARA(6) indicates that at a low temperature (1 degrees C) the MEARA(6) peptide has a relatively high helical content (76% at pH 2.0 and 65% at pH 7.0). The effects of pH and ionic strength on the CD spectrum of MEARA(6) suggest that a number of electrostatic interactions (e.g., i, i + 3 Arg/Glu ion pair, charge-dipole interactions) contribute to the stability of the helical structure in this peptide. DSC profiles show that the melting of MEARA(6) helix is accompanied by positive change in the enthalpy. To determine thermodynamic parameters of helix-coil transition from DSC profiles for this peptide, we developed a new, semiempirical procedure based on the calculated function for the heat capacity of the coiled state for a broad temperature range. The application of this approach to the partial molar heat capacity function for MEARA(6) provides the enthalpy change for helix formation calculated per amino acid residue as 3.5 kJ/mol.

Structural and functional study of a conserved region in the uncoupling protein UCP1: the three matrix loops are involved in the control of transport

It has been reported that the region 261-269 of the uncoupling protein from brown adipose tissue mitochondria, UCP1, has an important role in the control of its proton translocating activity. Thus the deletion of residues Phe267-Lys268-Gly269 leads to the loss of the nucleotide regulation of the protein, while the complete deletion of the segment leads to the formation of a pore. The region displays sequence homology with the DNA-binding domain of the estrogen receptor. The present report analyzes the structure, by NMR and circular dichroism, of a 20 amino acid residue peptide containing the residues of interest. We demonstrate that residues 263-268 adopt an α-helical structure. The helix is at the N-terminal end of the sixth transmembrane domain. The functional significance of this helix has been examined by site-directed mutagenesis of the protein expressed recombinantly in yeasts. Alterations in the structure or orientation of the region leads to an impairment of the regulation, by nucleotides and fatty acids, of the transport activity. UCP1 is one member of the family formed by the carriers of the mitochondrial inner membrane. The family is characterized by a tripartite structure with three repeated segments of about 100 amino acid residues. Two of the mutations have also been performed in the first and second matrix loops and the effect on UCP1 function is very similar. We conclude that the three matrix loops contribute to the formation of the gating domain in UCP1 and propose that they form a hydrophobic pocket that accommodates the purine moiety of the bound nucleotide.

CD and NMR investigations on trifluoroethanol-induced step-wise folding of helical segment from scorpion neurotoxin.

A 14 amino acid residue peptide from the helical region of Scorpion neurotoxin has been structurally characterized using CD and NMR spectroscopy in different solvent conditions. 2,2,2-Trifluoroethanol (TFE) titration has been carried out in 11 steps from 0 to 90% TFE and the gradual stabilization of the conformation to form predominantly alpha-helix covering all of the 14 residues has been studied by 1H and 13C NMR spectroscopy. Detailed information such as coupling constants, chemical shift indices, NOESY peak intensities and amide proton temperature coefficients at each TFE concentration has been extracted and analysed to derive the step-wise preferential stabilization of the helical segments along the length of the peptide. It was found that there is a finite amount of the helical conformation in the middle residues 5-11 even at low TFE concentrations. It was also observed that > 75% TFE (v/v) is required for the propagation of the helix to the N and C termini and for correct packing of the side chains of all of the residues. These observations are significant to understanding the folding of this segment in the protein and may throw light on the inherent preferences and side chain interactions in the formation of the helix in the peptide.

Millisecond radiolytic modification of peptides by synchrotron X-rays identified by mass spectrometry.

Radiolysis of peptide and protein solutions with high-energy X-ray beams induces stable, covalent modifications of amino acid residues that are useful for synchrotron protein footprinting. A series of 5-14 amino acid residue peptides of varied sequences were selected to study their synchrotron radiolysis chemistry. Radiolyzed peptide products were detected within 10 ms of exposure to a white light synchrotron X-ray beam. Mass spectrometry techniques were used to characterize radiolytic modification to amino acids cysteine (Cys), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), proline (Pro), histidine (His), and leucine (Leu). A reactivity order of Cys, Met >> Phe, Tyr, > Trp > Pro > His, Leu was determined under aerobic reaction conditions from MS/MS analysis of the radiolyzed peptide products. Radiolysis of peptides in 18O-labeled water under aerobic conditions revealed that oxygenated radical species from air and water both contribute to the modification of amino acid side chains. Cysteine and methionine side chains reacted with hydroxyl radicals generated from radiolysis of water as well as molecular oxygen. Phenylalanine and tyrosine residues were modified predominantly by hydroxyl radicals, and the source of modification of proline was exclusively through molecular oxygen.

Selection of a Peptide Ligand to the p75 Neurotrophin Receptor Death Domain and Determination of Its Binding Sites by NMR

The p75 neurotrophin receptor (p75NTR) contains a conserved death domain module similar to that of the cytotoxic receptors Fas and TNFR-1. Here, we describe the selection of peptide ligands from a combinatorial library using a variation of the selectively-infective phage (SIP) method directed to the death domain of p75NTR. The binding sites on the death domain of p75NTRwere identified for a 15 amino acid residue peptide by nuclear magnetic resonance (NMR) spectroscopy. The selected peptides may be useful for probing the function of the p75NTRdeath domain and aid in defining its downstream signalling mechanism.