Synergistic inhibitory activity of alpha- and beta-LFA-1 peptides on LFA-1/ICAM-1 interaction.

Interactions of cell-adhesion molecule LFA-1 and its ligand ICAM-1 play important roles during immune and inflammatory responses. Critical residues of LFA-1 for ICAM-1 binding are known to be in the I-domain of the alpha-subunit and the I-like domain of the beta-subunit. On the basis of our previous work demonstrating the inhibitory activity of I-domain cyclic peptide cLAB.L on LFA-1/ICAM-1 interaction, here we have explored the activity of I-like-domain peptide LBE on the binding mechanism of cLAB.L. LBE enhances cLAB.L binding to T-cells and epithelial cells. The adherence of T-cells to epithelial monolayers was suppressed by the two peptides. The addition of LBE to the monolayers prior to the addition cLAB.L produced a better inhibitory effect than the reverse procedure. LBE, but not cLAB.L, changes the ICAM-1 conformation, suggesting that LBE binds to ICAM-1 at sites that are distinct from these of cLAB.L and induces improved conformation in ICAM-1 for binding to cLAB.L.     

A Ca2+ binding cyclic peptide derived from the alpha-subunit of LFA-1: inhibitor of ICAM-1/LFA-1-mediated T-cell adhesion.

The objective of this work is to study the conformation of cyclic peptide (1), cyclo (1, 12) Pen1-Gly2-Val3-Asp4-Val5-Asp6-Gln7-+ ++Asp8-Gly9-Glu10-Thr11-Cys12, in the presence and absence of calcium. Cyclic peptide 1 is derived from the divalent cation binding sequence of the alpha-subunit of LFA-1. This peptide has been shown to inhibit ICAM-1-LFA-1 mediated T-cell adhesion. In order to understand the structural requirements for this biologically active peptide, its solution structure was studied by nuclear magnetic resonance (NMR), circular dichroism (CD) and molecular dynamics simulations. This cyclic peptide exhibits two types of possible conformations in solution. Structure I is a loop-turn-loop type of structure, which is suitable to bind cations such as EF hand proteins. Structure II is a more extended structure with beta-hairpin bend at Asp4-Val5-Asp6-Gln7. There is evidence that alterations in the conformation of LFA-1 upon binding to divalent cations cause LFA-1 to bind to ICAM-1. To understand this mechanism, the cation-binding properties of the peptide were studied by CD and NMR. CD studies indicated that the peptide binds to calcium and forms a 1 : 1 (peptide: calcium) complex at low calcium concentrations and multiple types of complexes at higher cation concentrations. NMR studies indicated that the conformation of the peptide is not significantly altered upon binding to calcium. The peptide can inhibit T-cell adhesion by directly binding to ICAM-1 or by disrupting the interaction of the alpha and beta-subunits of LFA-1 protein. This study will help us to understand the mechanism(s) of action of this peptide and will improve our ability to design a better inhibitor of T-cell adhesion.     

Synergistic inhibitory activity of α- and β-LFA-1 peptides on LFA-1/ICAM-1 interaction

Interactions of cell-adhesion molecule LFA-1 and its ligand ICAM-1 play important roles during immune and inflammatory responses. Critical residues of LFA-1 for ICAM-1 binding are known to be in the I-domain of the α-subunit and the I-like domain of the β-subunit. On the basis of our previous work demonstrating the inhibitory activity of I-domain cyclic peptide cLAB.L on LFA-1/ICAM-1 interaction, here we have explored the activity of I-like-domain peptide LBE on the binding mechanism of cLAB.L. LBE enhances cLAB.L binding to T-cells and epithelial cells. The adherence of T-cells to epithelial monolayers was suppressed by the two peptides. The addition of LBE to the monolayers prior to the addition cLAB.L produced a better inhibitory effect than the reverse procedure. LBE, but not cLAB.L, changes the ICAM-1 conformation, suggesting that LBE binds to ICAM-1 at sites that are distinct from these of cLAB.L and induces improved conformation in ICAM-1 for binding to cLAB.L.     

A peptide derived from LFA-1 protein that modulates T-cell adhesion binds to soluble ICAM-1 protein

Leukocyte function associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) have been shown to be critical for adhesion process and immune response. Modulation or inhibition of the interaction between LFA-1/ICAM-1 interactions can result in therapeutic effects. Our group and others have shown that peptides derived from ICAM-1 or LFA-1 inhibit adhesion in a homotypic T-cell adhesion assay. It is likely that the peptides derived from ICAM-1 bind to LFA-1 and peptides derived from LFA-1 bind to ICAM-1 and inhibit the adhesion interaction. However, there are no concrete experimental evidence to show that peptides bind to either LFA-1 or ICAM-1 and inhibit the adhesion. Using NMR, CD and docking studies we have shown that an LFA-1 derived peptide binds to soluble ICAM-1. Docking studies using "autodock" resulted in LFA-1 peptide interacting with the ICAM-1 protein near Glu34. The proposed model based on our experimental data indicated that the LFA-1 peptide interacts with the protein via three intermolecular hydrogen bonds. Hydrophobic interactions also play a role in stabilizing the complex.     

A Peptide Derived from LFA-1 Protein that Modulates T-cell Adhesion Binds to Soluble ICAM-1 Protein

Abstract

Leukocyte function associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) have been shown to be critical for adhesion process and immune response. Modulation or inhibition of the interaction between LFA-1/ICAM-1 interactions can result in therapeutic effects. Our group and others have shown that peptides derived from ICAM- 1 or LFA-1 inhibit adhesion in a homotypic T-cell adhesion assay. It is likely that the peptides derived from ICAM-1 bind to LFA-1 and peptides derived from LFA-1 bind to ICAM- 1 and inhibit the adhesion interaction. However, there are no concrete experimental evidence to show that peptides bind to either LFA-1 or ICAM-1 and inhibit the adhesion. Using NMR, CD and docking studies we have shown that an LFA-1 derived peptide binds to soluble ICAM-1. Docking studies using “autodock” resulted in LFA-1 peptide interacting with the ICAM-1 protein near Glu34. The proposed model based on our experimental data indicated that the LFA-1 peptide interacts with the protein via three intermolecular hydrogen bonds. Hydrophobic interactions also play a role in stabilizing the complex.     

Structural recognition of an ICAM-1 peptide by its receptor on the surface of T cells: conformational studies of cyclo (1, 12)-Pen-Pro-Arg-Gly-Gly-Ser-Val-Leu-Val-Thr-Gly-Cys-OH.

The purpose of this study is to elucidate the solution conformation of cyclic peptide 1 (cIBR), cyclo (1, 12)-Pen1-Pro2-Arg3-Gly4-Gly5-Ser6-Val7-Leu8-V al9-Thr10-Gly11-Cys12-OH, using NMR, circular dichroism (CD) and molecular dynamics (MD) simulation experiments. cIBR peptide (1), which is derived from the sequence of intercellular adhesion molecule-1 (ICAM-1, CD54), inhibits homotypic T-cell adhesion in vitro. The peptide hinders T-cell adhesion by inhibiting the leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18) interaction with ICAM-1. Furthermore, Molt-3 T cells bind and internalize this peptide via cell surface receptors such as LFA-1. Peptide internalization by the LFA-1 receptor is one possible mechanism of inhibition of T-cell adhesion. The recognition of the peptide by LFA-1 is due to its sequence and conformation; therefore, this study can provide a better understanding for the conformational requirement of peptide-receptor interactions. The solution structure of 1 was determined using NMR, CD and MD simulation in aqueous solution. NMR showed a major and a minor conformer due to the presence of cis/trans isomerization at the X-Pro peptide bond. Because the contribution of the minor conformer is very small, this work is focused only on the major conformer. In solution, the major conformer shows a trans-configuration at the Pen1-Pro2 peptide bond as determined by HMQC NMR. The major conformer shows possible beta-turns at Pro2-Arg3-Gly4-Gly5, Gly5-Ser6-Val7-Leu8, and Val9-Thr10-Gly11-Cys12. The first beta-turn is supported by the ROE connectivities between the NH of Gly4 and the NH of Gly5. The connectivities between the NH of Ser6 and the NH of Val7, followed by the interaction between the amide protons of Val7 and Leu8, support the presence of the second beta-turn. Furthermore, the presence of a beta-turn at Val9-Thr10-Gly11-Cys12 is supported by the NH-NH connectivities between Thr10 and Gly11 and between Gly11 and Cys12. The propensity to form a type I beta-turn structure is also supported by CD spectral analysis. The cIBR peptide (1) shows structural similarity at residues Pro2 to Val7 with the same sequence in the X-ray structure of D1-domain of ICAM-1. The conformation of Pro2 to Val7 in this peptide may be important for its binding selectivity to the LFA-1 receptor.     

Structural and ICAM-1-docking properties of a cyclic peptide from the I-domain of LFA-1: an inhibitor of ICAM-1/LFA- 1-mediated T-cell adhesion

The purpose of this work was to study the conformation of cyclic peptide 1, cyclo(1,12)-Pen1-Ile2-Thr3-Asp4-Gly5-Glu6-Ala7- Thr8-Asp9-Ser10-Gly11-Cys12-OH, derived from the I-domain of the LFA-1 alpha-subunit. We found that cyclic peptide 1 can bind to the D1-domain of ICAM-1 and inhibit ICAM-1/LFA-1-mediated homotypic and heterotypic T-cell adhesion. To understand the bioactive conformation and binding requirements for cyclic peptide 1, its solution structure was studied using NMR, CD, and molecular dynamics simulations. Furthermore, possible binding properties between the cyclic peptide and the D1-domain of ICAM-1 were evaluated using docking experiments. This cyclic peptide has a stable betaII -turn at Asp4- Gly5-Glu6-Ala7 and a betaI-turn at Pen1-Ile2-Thr3-Asp4; a less stable betaV-turn is found at the C-terminal region. The beta-turn at Asp4- Gly5-Glu6-Ala7 was also found in the X-ray structure of the I-domain of LFA-1. Our CD studies showed that the peptide binds to calcium/magnesium and forms a 1:1 (peptide:calcium/magnesium) complex with low cation concentrations and multiple types of complexes with higher cation concentrations. Binding to divalent cations causes a conformational change in peptide 1; this is consistent with our previous study that binding of peptide 1 to ICAM-1 was influenced by divalent cations. Docking studies show the interaction between cyclic peptide 1 and the D1-domain of ICAM-1; it indicates that the Ile2-Thr3-Asp4-Gly4-Glu6-Ala7-Thr8 sequence interacts with the F and C strands of the D1-domain. Finally, these studies will help us design a new generation of selective peptides that may bind better to the D1-domain of ICAM-1.     

Targeting ICAM-1/LFA-1 interaction for controlling autoimmune diseases: designing peptide and small molecule inhibitors

This review describes the role of modulation of intracellular adhesion molecule-1 (ICAM-1)/leukocyte function-associated antigen-1 (LFA-1) interaction in controlling autoimmune diseases or inducing immunotolerance. ICAM-1/LFA-1 interaction is essential for T-cell activation as well as for migration of T-cells to target tissues. This interaction also functions, along with Signal-1, as a co-stimulatory signal (Signal-2) for T-cell activation, which is delivered by the T-cell receptors (TCR)-major histocompatibility complex (MHC)-peptide complex. Therefore, blocking ICAM-1/LFA-1 interaction can suppress T-cell activation in autoimmune diseases and organ transplantation. Many types of inhibitors (i.e. antibodies, peptides, small molecules) have been developed to block ICAM-1/LFA-1 interactions, and some of these molecules have reached clinical trials. Peptides derived from ICAM-1 and LFA-1 sequences have been shown to inhibit T-cell adhesion and activation. In addition, these inhibitors have been useful in elucidating the mechanism of ICAM-1/LFA-1 interaction. Besides binding to LFA-1, the ICAM-1 peptide can be internalized by LFA-1 receptors into the cytoplasmic domain of T-cells. Therefore, this ICAM-1 peptide can be utilized to selectively target toxic drugs to T-cells, thus avoiding harmful side effects. Finally, bi-functional inhibitory peptide (BPI), which is made by conjugating the antigenic peptide and an LFA-1 peptide, can alter the T-cell commitment from T-helper-1 (Th1) to T-helper-2 (Th2)-like cells, suggesting that this peptide may have a role in blocking the formation of the "immunological synapse."     

Conformational properties of five peptides corresponding to the entire sequence of glutathione transferase domain II

Five peptides matching the helices alpha4, alpha5, alpha6, alpha7, and alpha8, spanning the entire sequence of domain II of pG-STP1-1, have been synthesized and their conformations analyzed by far-UV CD spectroscopy. The results show that a5, a7, and a8 peptides are unstructured in water/2,2,2-trifluoroethanol (TFE) solutions. The a4-peptide also adopts random conformations in aqueous solvent. Moreover, the relative low helical content (20%), estimated for this peptide in the presence of 30% (v/v) TFE, suggests that the sequence of this protein fragment does not possess sufficient information for a strong helical propensity. On the contrary, the synthesized a6 peptide, in the presence of TFE, showed a relevant structural autonomy with a helical content (41%) which was significantly higher than that estimated, under the same conditions, for all other peptides. More in general in the presence of solvents less polar than water, the isolated a6 peptide shows the same helical conformation adopted by the corresponding alpha6-helix in the hydrophobic core of the protein. A n-capping box motif, strictly conserved at the N-terminal of the alpha6-helix of all GST and related protein including eucaryotic translation elongation factor (EF1gamma) and the yeast prion protein Ure2, plays an important role in the alpha-helix nucleation and stability of this protein fragment. The results suggest that the alpha6-helix might represent a nucleation site of GST folding and that the helical conformation of this region of the protein is an important requirement during earlier events of GST refolding.     

Structural and ICAM-1-Docking Properties of a Cyclic Peptide from the I-domain of LFA-1: An inhibitor of ICAM-1/LFA-1-mediated T-cell adhesion

Abstract

The purpose of this work was to study the conformation of cyclic peptide 1, cyclo(1,12)- Pen1-Ile2-Thr3-Asp4-Gly5-Glu6-Ala7-Thr8-Asp9-Ser10-Gly11-Cys12-OH, derived from the I-domain of the LFA-1 α-subunit. We found that cyclic peptide 1 can bind to the D1- domain of ICAM-1 and inhibit ICAM-1/LFA-1-mediated homotypic and heterotypic T-cell adhesion. To understand the bioactive conformation and binding requirements for cyclic peptide 1, its solution structure was studied using NMR, CD, and molecular dynamics simulations. Furthermore, possible binding properties between the cyclic peptide and the D1- domain of ICAM-1 were evaluated using docking experiments. This cyclic peptide has a stable βII'-turn at Asp4-Gly5-Glu6-Ala7 and a βI-turn at Pen1-Ile2-Thr3-Asp4; a less stable βV-turn is found at the C-terminal region. The β-turn at Asp4-Gly5-Glu6-Ala7 was also found in the X-ray structure of the I-domain of LFA-1. Our CD studies showed that the peptide binds to calcium/magnesium and forms a 1:1 (peptide:calcium/magnesium) complex with low cation concentrations and multiple types of complexes with higher cation concentrations. Binding to divalent cations causes a conformational change in peptide 1; this is consistent with our previous study that binding of peptide 1 to ICAM-1 was influenced by divalent cations. Docking studies show the interaction between cyclic peptide 1 and the D1- domain of ICAM-1; it indicates that the Ile2-Thr3-Asp4-Gly4-Glu6-Ala7-Thr8 sequence interacts with the F and C strands of the D1-domain. Finally, these studies will help us design a new generation of selective peptides that may bind better to the D1-domain of ICAM-1.     

Solution structure of humanin, a peptide against Alzheimer's disease-related neurotoxicity

Humanin is a newly identified 24-residue peptide that suppresses neuronal cell death caused by a wide spectrum of familial Alzheimer's disease genes and the beta-amyloid peptide. In this study, NMR and circular dichroism studies of synthetic humanin in aqueous and 30% 2,2,2-trifluoroethanol (TFE) solutions are reported. In aqueous solution, humanin exists predominantly in an unstructured conformation in equilibrium with turn-like structures involving residues Gly5 to Leu10 and Glu15 to Leu18, providing indication of nascent helix. In the less polar environment of 30% TFE, humanin readily adopts helical structure with long-range order spanning residues Gly5 to Leu18. Comparative 3D modeling studies and topology predictions are in qualitative agreement with the experimental findings in both environments. Our studies reveal a flexible peptide in aqueous environment, which is free to interact with possible receptors that mediate its action, but may also acquire a helical conformation necessary for specific interactions and/or passage through membranes.     

Inhibition of ICAM-1/LFA-1-mediated heterotypic T-cell adhesion to epithelial cells: design of ICAM-1 cyclic peptides

In this work, we have designed cyclic peptides (cIBL, cIBR, cIBC, CH4 and CH7) derived from the parent IB peptide (ICAM-1(1-21)) that are inhibitors of ICAM-1/LFA-1-mediated T-cell adhesion to Caco-2 cell monolayers. Cyclic peptide cIBR has the best activity of any of the peptides evaluated. The active ICAM-1 peptides have a common Pro-Arg-Gly sequence that may be important for binding to LFA-1.     

Spectroscopic studies of a phosphoinositide-binding peptide from gelsolin: behavior in solutions of mixed solvent and anionic micelles.

The peptide G(150-169) corresponds to a phosphatidylinositol 4,5-bisphosphate (PIP2) and filamentous actin (F-actin) binding site on gelsolin (residues 150-169, with the sequence KHVVPNEVVVQRLFQVKGRR). The conformation of this peptide in trifluoroethanol (TFE) aqueous solution was determined by 1H nuclear magnetic resonance as the first step toward understanding the structural aspects of the interaction of G(150-169) and PIP2. The circular dichroism experiments show that G(150-169) adopts a predominantly alpha-helical form in both 50% TFE aqueous solution and in the presence of PIP2 micelles, therefore establishing a connection between the two conformations. 1H nuclear magnetic resonance experiments of G(150-169) in TFE co-solvent show that the helical region extends from Pro-154 to Lys-166. The amphiphilic nature of this helical structure may be the key to understanding the binding of the peptide to lipids. Sodium dodecyl sulfate micelle solution is used as a model for anionic lipid environments. Preliminary studies of the conformation of G(150-169) in sodium dodecyl sulfate micelle solution show that the peptide forms an alpha-helix similar to but with some structural differences from that in TFE co-solvent. Fluorescence experiments provide evidence of peptide clustering over a narrow range of peptide/PIP2 ratios, which is potentially relevant to the biological function of PIP2.     

Conformations of hydrophobic peptides in trifluoroethanol, water and in solid state: a circular dichroism and Fourier Transform Infrared study

The conformations of peptides corresponding to KLLIALVLCFLPLAALG have been examined in trifluoroethanol (TFE), aqueous medium by circular dichroism spectroscopy and in the solid state by Fourier Transform Infra Red Spectroscopy (FTIR). The 17-residue parent peptide and peptides corresponding to shorter segments LVLCFLPLAALG and CFLPLAALG showed preference for helical conformation in TFE. Even the shorter hydrophobic peptides corresponding to KLLIA and LVL showed propensity for beta-turn conformations in TFE. However, peptides corresponding to the relatively polar segment FLPLAALG were unordered in TFE. In water, peptides that showed ordered conformation in TFE preferred beta-conformation. In solid-state, FTIR spectra indicated that the hydrophobic peptides adopt beta-structures with extensive hydrogen bonded network in the solid-state. The hydrophobic core segment thus appears to dictate the conformational propensity of the peptide.     

Hints of nonhierarchical folding of acidic fibroblast growth factor

We have analyzed by circular dichroism (CD) and proton nuclear magnetic resonance (NMR) the helical propensity of the all-beta protein acidic fibroblast growth factor (aFGF) and two peptides corresponding to beta-strand 8 (beta8 peptide, amino acids 95-107) and the beta-strand 8/turn/beta-strand 9 hairpin (beta8/9 peptide, amino acids 95-114), which has been involved in receptor binding. A secondary structure prediction of aFGF carried out by several procedures labels the 95-104 sequence as predominantly alpha-helical. A titration of aFGF with 2,2,2-trifluoroethanol (TFE) induces a change in the far-UV CD spectrum of the protein giving rise to a prominent alpha-helical shape (22% alpha-helix). The cooperativity of the transition and the moderate TFE concentrations used (midpoint at 24%) suggest that the effect of TFE is specific. Moreover, a titration performed at pH 2 yields a higher amount of alpha-helix (55%) at a smaller TFE concentration. Synthetic peptides containing the beta8 and beta8/9 sequences display a random coil conformation at pH 7 but acquire alpha-helical structure in the presence of TFE, methanol, and SDS micelles. At pH below 3.0 a significant amount (20-30%) of alpha-helical conformation is present in both the beta8 and beta8/9 peptides even in the absence of other solvent additives. The secondary structure of the peptides was determined by proton nuclear magnetic resonance (1H NMR). These results suggest that the 95-114 sequence of aFGF has helical propensity and that the protein may fold nonhierarchically in the early steps of folding, acquiring its final beta-structure by a later interaction with the rest of the polypeptide.     

Conformational solution studies of neuropeptide gamma using CD and NMR spectroscopy.

Neuropeptide gamma is one of the largest members of the tachykinin family of peptides, exhibiting strong agonistic activity towards the NK-2 tachykinin receptor. This peptide was synthesized by the solid-phase method using the Fmoc chemistry. Circular-dichroism spectroscopy (CD) investigations of this peptide were performed in phosphate buffer, in the presence of sodium dodecylsulphate (SDS) micelles and trifluoroethanol (TFE) solutions and in DMSO-d6 using the 2D NMR technique in conjunction with two different theoretical approaches. The first assumes multiconformational equilibrium of the peptide studied characterized by the values of statistical weights of low-energy conformations. These calculations were performed using three different force fields ECEPP/3, AMBER4.1 and CHARMM (implemented in the X-PLOR program). The second method incorporates interproton distance and dihedral angle constraints into the starting conformation using the Simulated Annealing algorithm (X-PLOR program). The CD experiments revealed that although the peptide studied is flexible in polar solvents, a tendency to adopt a helical structure was observed in the hydrophobic environment. The NMR data (NOE effects) indicate a helical or reverse structure in the Ile7-His12 fragment of the peptide studied in DMSO-d6 solution. The results obtained cannot be interpreted in terms of a single conformation. Most of the conformations obtained with the ECEPP/3 force field possess a high content of a helical structure. None of the conformers, obtained with the AMBER4.1 and CHARMM force fields, can be considered as the dominant one. In all conformations several beta-turns were detected and in some cases gamma-turns were also found. But in fact, it is rather difficult to select the position of the secondary element(s) present in the structure of NPgamma in solution. All conformers calculated with the X-PLOR program (with using NMR derived distance and torsion angle constraints) are stabilized by several beta-turns. Common structural motives are a type IV beta-turn in the Gln6-His12 fragment. All conformations obtained using two approaches adopt very similar turn shapes in the middle region of molecule and a random structure on the N- and C-terminal fragments.     

Two peptide fragments G55-I72 and K97-A109 from staphylococcal nuclease exhibit different behaviors in conformational preferences for helix formation

Two synthetic peptides, SNasealpha1 and SNasealpha2, corresponding to residues G55-I72 and K97-A109, respectively, of staphylococcal nuclease (SNase), are adopted for detecting the role of helix alpha1 (E57-A69) and helix alpha2 (M98-Q106) in the initiation of folding of SNase. The helix-forming tendencies of the two SNase peptide fragments are investigated using circular dichroism (CD) and two-dimensional (2D) nuclear magnetic resonance (NMR) methods in water and 40% trifluoroethanol (TFE) solutions. The coil-helix conformational transitions of the two peptides in the TFE-H2O mixture are different from each other. SNasealpha1 adopts a low population of localized helical conformation in water, and shows a gradual transition to helical conformation with increasing concentrations of TFE. SNasealpha2 is essentially unstructured in water, but undergoes a cooperative transition to a predominantly helical conformation at high TFE concentrations. Using the NMR data obtained in the presence of 40% TFE, an ensemble of alpha-helical structures has been calculated for both peptides in the absence of tertiary interactions. Analysis of all the experimental data available indicates that formation of ordered alpha-helical structures in the segments E57-A69 and M98-Q106 of SNase may require nonlocal interactions through transient contact with hydrophobic residues in other parts of the protein to stabilize the helical conformations in the folding. The folding of helix alpha1 is supposed to be effective in initiating protein folding. The formation of helix alpha2 depends strongly on the hydrophobic environment created in the protein folding, and is more important in the stabilization of the tertiary conformation of SNase.     

Solution conformation of a peptide corresponding to the principal neutralizing determinant of HIV-1IIIB: a two-dimensional NMR study.

The 24 amino acid peptide RP135 corresponds in its amino acid sequence to the principal neutralizing determinant (PND) of the IIIB isolate of HIV-1. Although the sequence of the PND is highly variable, its central part, containing the sequence GPGR, is conserved in most HIV isolates. Using 2D NMR and CD spectroscopy, we have studied the conformation of RP135 and of two shorter versions: one (P547) that includes the GPGR sequence with the N-terminal part of the peptide and the other (P344) that includes GPGR and the C-terminal segment of RP135. In water, the C-terminal part of RP135 was found to exist in several transient turnlike conformations ("nascent helix"). A helical conformation was found to be stabilized by the addition of TFE. A transient turn was observed also in the GPGR sequence, both in water and in aqueous TFE solutions. While no nascent helix conformations could be observed in the N-terminal part of RP135 in water, a helical conformation was partially stabilized by the addition of TFE. The conformations of the two shorter versions of the peptide were similar to those of the corresponding parts of RP135, except that the transient turn in GPGR could not be detected in P547 dissolved in water. The turn in GPGR was previously predicted by Larosa et al. (1990) and was observed by Chandrasekhar et al. (1991) in the PND peptide of HIV-1MN (RP142), which shares only 56% identity with RP135. However, nascent helix conformations were not observed in aqueous solutions of RP142.(ABSTRACT TRUNCATED AT 250 WORDS)     

Context-dependent conformation of diethylglycine residues in peptides.

Diethylglycine (Deg) residues incorporated into peptides can stabilize fully extended (C5) or helical conformations. The conformations of three tetrapeptides Boc-Xxx-Deg-Xxx-Deg-OMe (Xxx=Gly, GD4; Leu, LD4 and Pro, PD4) have been investigated by NMR. In the Gly and Leu peptides, NOE data suggest that the local conformations at the Deg residues are fully extended. Low temperature coefficients for the Deg(2) and Deg(4) NH groups are consistent with their inaccessibility to solvent, in a C5 conformation. NMR evidence supports a folded beta-turn conformation involving Deg(2)-Gly(3), stabilized by a 4-->1 intramolecular hydrogen bond between Pro(1) CO and Deg(4) NH in the proline containing peptide (PD4). The crystal structure of GD4 reveals a hydrated multiple turn conformation with Gly(1)-Deg(2) adopting a distorted type II/II' conformation, while the Deg(2)-Pro(3) segment adopts a type III/III' structure. A lone water molecule is inserted into the potential 4-->1 hydrogen bond of the Gly(1)-Deg(2) beta-turn.     

Identification of initiation sites for T4 lysozyme folding using CD and NMR spectroscopy of peptide fragments

Using CD and 2D (1)H NMR spectroscopy, we have identified potential initiation sites for the folding of T4 lysozyme by examining the conformational preferences of peptide fragments corresponding to regions of secondary structure. CD spectropolarimetry showed most peptides were unstructured in water, but adopted partial helical conformations in TFE and SDS solution. This was also consistent with the (1)H NMR data which showed that the peptides were predominantly disordered in water, although in some cases, nascent or small populations of partially folded conformations could be detected. NOE patterns, coupling constants, and deviations from random coil Halpha chemical shift values complemented the CD data and confirmed that many of the peptides were helical in TFE and SDS micelles. In particular, the peptide corresponding to helix E in the native enzyme formed a well-defined helix in both TFE and SDS, indicating that helix E potentially forms an initiation site for T4 lysozyme folding. The data for the other peptides indicated that helices D, F, G, and H are dependent on tertiary interactions for their folding and/or stability. Overall, the results from this study, and those of our earlier studies, are in agreement with modeling and HD-deuterium exchange experiments, and support an hierarchical model of folding for T4 lysozyme.