A novel beta-turn location in an LHRH antagonist: a combined conformational search and molecular dynamics study

A 50 pico-second molecular dynamics simulation on a cyclic LHRH antagonist analogue Ac-D-Phe1-D-Phe2-D-Trp3-Ser4-Glu5-D-Arg6-Leu7-Lys8+ ++-Pro9-D-Ala10-NH2 (where the cyclisation is via an amide linkage between the Glu5 and Lys8 side chains), reveals some hitherto unseen conformational features. The LHRH analogue is found to adopt a near beta-sheet type of conformation with the reversal in the chain being brought about by a D-Trp3-Ser4-Glu5-D-Arg6 beta turn. The N- and C-terminal ends of the peptide come close together and interact through a network of hydrogen bonds. Additional hydrogen bonds expected of a sheet type of conformation stabilise the lowest energy minima. A conformational search of all possible cyclic structures of a model system c(Glu-D-Ala-Ala-Lys) which was used to determine the starting structure for the simulation studies of the cyclic LHRH antagonist analogue is also highlighted. The influence of the cyclic part on the conformation of this LHRH analogue is discussed.     

Structural characterization of lipopeptide agonists for the bradykinin B2 receptor

The conformational features of Pam-Lys(0)-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH (PKD) and Pam-Gly(-1)-Lys(0)-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH (PGKD), the Pam-Lys and Pam-Gly-Lys analogues of bradykinin, have been determined by high-resolution NMR in a zwitterionic lipoid environment. Radical-induced relaxation of the (1)H NMR signals was used to probe the topological orientation of the peptides with respect to the zwitterionic lipid interface. The radical-induced relaxation and molecular dynamics (MD) data indicated that the palmitic acid and N-terminal amino acid residues embed into the micelles, while the rest of the polypeptide chain is closely associated with the water-micelle interface. Throughout the entire nuclear Overhauser effect restrained MD simulation, a nonideal type I beta-turn was observed in the C-terminus of PKD between residues 6 and 9, and a gamma-turn was observed in the C-terminus of PGKD between residues 6 and 7. Therefore, the additional glycine has a dramatic effect on the structural preferences of the biologically important C-terminus, an effect brought about by the interaction with the lipid environment. These structural features are correlated to the biological activity at the bradykinin B2 receptor.     

Two conformational states of Turkey ovomucoid third domain at low pH: three-dimensional structures,internal dynamics,and interconversion kinetics and thermodynamics

Turkey ovomucoid third domain (OMTKY3) is shown to exist at low pH as two distinctly folded, interconverting conformations. Activation parameters were determined for the transition, and these were of the type reported previously for cis/trans isomerizations of prolyl peptide bonds. Multidimensional, multinuclear NMR spectroscopy was used to determine the three-dimensional structure of each of the two states of P(5)-Pro(14)Asp OMTKY3 at pH 2.5 and 25 degrees C, under conditions where the two states have equal populations with interchange rates of 0.25 s(-1). The results showed that the two states differ by cis/trans isomerization of the P(8)-Tyr(11)-P(7)-Pro(12) peptide bond, which is cis in the conformer dominant at neutral pH and trans in the conformer appearing at low pH. The major structural differences were found to be in the region of the reactive site loop. The core of the protein, including the antiparallel beta-sheet and a alpha-helix, is preserved in both structures. The state with the cis peptide bond is similar to previously reported structures of OMTKY3 determined by NMR spectroscopy and X-ray crystallography. The cis-to-trans transition results in the relocation of the aromatic ring of P(8)-Tyr(11), disrupts many interactions between the alpha-helix and the reactive-site loop, and leads to more open spacing between this loop and the alpha-helix. In addition, the configurations of two of the three disulfide bonds, P(11)-Cys(8)- P(20)'-Cys(38), and P(3)-Cys(16)- P(17)'-Cys(35), are altered such that the C(alpha)-C(alpha) distances for each disulfide bridge are longer by approximately 1 A in the trans state than in the cis. Mutations at P(1)-Leu(18), P(6)-Lys(13), and P(5)-Pro(14) influence the position of the cis <= => trans equilibrium. In P(1)-Leu(18)Xxx OMTKY3 mutants, the trans state is more favored by P(1)-Gly(18) than by Ala(18) or Leu(18); in P(6)-Lys(13)Xxx OMTKY3 mutants, the trans state is more favored by P(6)-Glu(13) and P(6)-Asp(13) than Lys(13) or His(13). Stabilization of the trans state in P(5)-Pro(14)Xxx OMTKY3 mutants follows the series Xxx = Gly > Asp > Glu > Ala approximately equal His > Pro. In comparing the state with the trans peptide bond to that with the cis, the pK(a) values of P(12)-Asp(7) and P(1)'-Glu(19) are higher and those of P(9)-Glu(10) and P(25)'-Glu(43) are lower. The pK(a) values of other titrating groups in the molecule are similar in both conformational states. These pK(a) changes underlie the pH dependence of the conformational equilibrium and can be explained in part by observed structural differences. (15)N transverse relaxation results indicate that residues P(6)-Lys(13)-P(3)-Cys(16) in the trans state undergo a dynamic process on the microsecond-millisecond time scale not present in the cis state.     

Conformational studies of cyclic enkephalin analogues with L- or D-proline in position 3.

The conformation of a series of cyclic enkephalin analogues of a general formula X(1)-cyclo[Y(2)-Z(3)-Nal(4)-Leu(5)] (Nal: beta-(2-naphthyl)alanine), where X = Tyr, Phe, or Phe(NO(2)), Y = D-Dab or L-Dab (Dab: 2,4-diaminobutyric acid), and Z = D-Pro or L-Pro, was studied by means of NMR spectroscopy and theoretical conformational analysis with the Empirical Conformational Energy Program for Peptides and Proteins force field plus solvation. The NMR measurements were performed in dimethyl sulfoxide solution. The nuclear Overhauser effect intensities and coupling constants were used to compute the statistical weights of the conformations of the ensemble generated in global conformational searches. The purpose of this study was to determine whether introducing the D- or L-proline residue in position 3 can produce peptides with both rigid backbone and significant separation of the pharmacophore groups in position 1 and 4 (as required for high affinity for the mu-type opioid receptors). It was found that the analogues with D-Dab in position 2 and D-Pro in position 3 possess a stable type II' beta-turn at positions 3 and 4, which rigidifies the cyclic backbone; this finding was confirmed by independent measurements of the temperature coefficients of the amide protons, which indicated very significant screening of the Leu(5) amide proton from the solvent. However, these analogues were found to possess a short interchromophore distance. The analogues containing both Dab and Pro in the L-configuration are characterized by a larger interchromophore distance; however, they do not possess a stable beta-turn and have therefore a higher conformational flexibility. The modifications proposed in this work are therefore not likely to lead to enkephalin analogues with a high affinity for the mu-receptors.     

Study of bradykinin conformation in the presence of model membrane by Nuclear Magnetic Resonance and molecular modelling

The conformation of bradykinin (BK), Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9, was investigated by Nuclear Magnetic Resonance (NMR) spectroscopy and Monte Carlo simulation in two different media, i.e. in pure aqueous solution and in the presence of phospholipid vesicles. Monolamellar liposomes are a good model for biological membranes and mimic the environment experienced by bradykinin when interacting with G-protein coupled receptors (GPCRs). The NMR spectra showed that lipid bilayers induced a secondary structure in the otherwise inherently flexible peptide. The results of ensemble calculations revealed conformational changes occurring rapidly on the NMR time scale and allowed for the identification of different families of conformations that were averaged to reproduce the NMR observables. These structural results supported the hypothesis of the central role played by the peptide C-terminal domain in biological environments, and provided an explanation for the different biological behaviours observed for bradykinin.     

Nuclear magnetic resonance analysis and conformational characterization of a cyclic decapeptide antagonist of gonadotropin-releasing hormone

Two-dimensional proton nuclear magnetic resonance spectroscopy at 500 MHz has been carried out on the cyclic decapeptide antagonist of gonadotropin-releasing hormone: cyclo-(delta 3-Pro1-D-pClPhe2-D-Trp3-Ser4-Tyr5-D-Trp6-NMeLeu7-Arg8- Pro9-beta-Ala 10). The antagonist exists in two slowly interconverting conformations. All data are consistent with the conclusion that one form has all-trans peptide bonds and the other has a cis beta-Ala10-delta3-Pro1 bond. With the use of sequential assignment methods, chemical shift assignments were obtained for all backbone and side-chain protons of both conformational isomers except for the serine and tyrosine hydroxyl groups and the C gamma, C delta, and guanidinium group protons of the arginine. Temperature dependence of spectral parameters and magnitudes of observed nuclear Overhauser effects support the interpretation that both conformers of the antagonist consist of two beta-turns (type II', D-Trp6-NMeLeu7; type II, delta 3-Pro1-D-pClPhe2) connected by extended antiparallel beta-like strands.     

Solution structure of a peptide derived from the beta subunit of LFA-1

Cell-adhesion molecules are critical for immune response. It is well known that the inhibition of adhesion is very effective in immunotherapy and that the peptides derived from leukocyte function associated antigen (LFA-1) and intercellular adhesion molecule (ICAM-1) modulate cell-adhesion interaction. The three-dimensional structure of a cyclic peptide, Cyclo(1,12)Pen(1)-Asp(2)-Leu(3)-Ser(4)-Tyr(5)-Ser(6)-Leu(7)-Asp(8)-Asp(9)-Leu(10)-Arg(11)-Cys(12) (cLBEL) derived from the beta subunit of LFA-1 which is known to modulate homotypic T-cell-adhesion process has been studied using NMR, CD and molecular dynamics (MD) simulation. The peptide exhibits two possible conformations in solution. Structure I has a conformation with two consecutive beta-turns involving residues Tyr(5)-Ser(6)-Leu(7)-Asp(8) and Asp(9)-Leu(10)-Arg(11)-Cys(12). Structure II has a beta-turn at Tyr(5)-Ser(6)-Leu(7)-Asp(8) and forms a beta-hairpin type of conformation.     

Structure investigation of amphiphilic cyclopeptides in isotropic and anisotropic environments-A model study simulating peptide-membrane interactions.

It has been proposed that the membrane allows a much more efficient binding of certain small or medium-sized amphiphilic messenger molecules to their receptor, not only by accumulation of the drug, but also by induction of orientations and conformations that are much more favorable for receptor docking than structures adopted in isotropic phases. A series of eight amphiphilic cyclic peptides containing lipophilic (L-alpha-aminodecanoic acid = Ada, L-alpha-aminohexadecanoic acid = Ahd, Nhdg = N-hexadecylglycine) and hydrophilic (Lys, Asp) amino acids were synthesized and examined by means of NMR spectroscopy and molecular dynamics (MD) simulations in isotropic (CDCl3) and membrane-mimicking anisotropic (SDS/H2O) solvents to study the influence of the environment on their individual conformations. NMR data of cyclo(-Gly1-D-Asp2-Ahd3-Ahd4-Asp5-Gly6+ ++-) (C4), cyclo(-Lys1-D-Pro2-Lys3-Ada4-Pro5-Ada6-) (C5) and cyclo(-Lys1-Pro2-Lys3-Ada4-D-Pro5-Ada6-) (C6) clearly indicate that those compounds are too rigid to perform a conformational change upon transition from an isotropic to an anisotropic environment. On the other hand, the experimental data of cyclo (-Gly1-Asp2-Ahd3-Ahd4-Asp5-Gly6-) (C1), cyclo(-Asp1-Ala2-Nhdg3-Ala4-D-Asp5-) (C7), and cyclo(-D-Asp1-Ala2-Nhdg3-Ala4-Asp5-) (C8) suggest highly flexible unstructured molecules in both environments. However, for cyclo(-Asp1-Asp2-Gly3-Ahd4-Ahd5-Gly6-) (C2) we observed a structure inducing effect of a membrane-like environment. The compound populates three different conformations in SDS/H2O, whereas in CDCI3 no preferred conformation can be detected. cyclo(-D-Asp1-Asp2-Gly3-Ahd4-Ahd5-Gly6-) (C3) clearly exhibits two different conformations with a shifted beta,beta-turn motif in CDCI3 and SDS/H2O solutions. The conformational change could be reproduced in a restraint-free MD simulation using the biphasic membrane mimetic CCl4/H2O. Our results give clear evidence that membrane interactions may not only lead to structure inductions, but can also induce major conformational changes in compounds already exhibiting a defined structure in isotropic solution.     

Searching for folding initiation sites of staphylococcal nuclease: a study of N-terminal short fragments

The N-terminal short fragments of staphylococcal nuclease (SNase), SNase20, SNase28, and SNase36, corresponding to the sequence regions, Ala1-Gly20, Ala1-Lys28, and Ala1-Leu36, respectively, as well as an 8-residue peptide (Ala17-Ile18-Asp19-Gly20-Asp21-Thr22-Val23-Lys24) have been synthesized. The conformational states of these fragments were investigated using CD and NMR spectroscopy in aqueous solution and in trifluoroethanol (TFE)-H(2)O mixture. SNase20 containing a sequence corresponding to a bent peptide in native SNase shows a transient population of bend-like conformation around Ala12-Thr13-Leu14 in TFE-H(2)O mixture. The sequence region of Ala17-Thr22 of SNase28 displays a localized propensity for turn-like conformation in both aqueous solution and TFE-H(2)O mixture. The conformational ensemble of SNase36 in aqueous solution includes populated turn-like conformations localized in sequence regions Ala17-Thr22 and Tyr27-Gln30. The analysis suggests that these sequence regions, which form the regular secondary structures in native protein, may serve as the folding nucleation sites of SNase fragments of different chain lengths starting from the N-terminal end. Thus, the formation of bend- and turn-like conformations of these sequence regions may be involved in the early folding events of the SNase polypeptide chain in vitro.     

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.     

Beta-turns induced in bradykinin by (S)-alpha-methylproline.

The ability of (S)-alpha-methylproline (alpha-MePro) to stabilise reverse-turn conformations in the peptide hormone bradykinin (BK = Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) has been investigated. Two BK analogues containing alpha-MePro at position 3 or position 7 were synthesised and their conformations in aqueous solution investigated by NMR spectroscopy. Whereas BK is largely disordered on the NMR time scale both analogues showed ROE connectivities in 2D-ROESY spectra indicative of reverse-turn conformations at both Pro2-Phe5 and Ser6-Arg9, whose formation appears to be cooperative. Some potential applications of alpha-MePro as a reverse-turn mimetic in the construction of synthetic peptide libraries is discussed.     

Analogs of alpha-melanocyte stimulating hormone with high agonist potency and selectivity at human melanocortin receptor 1b: the role of Trp(9) in molecular recognition

alpha-Melanocyte stimulating hormone (alphaMSH), Ac-Ser(1)-Tyr(2)-Ser(3)-Met(4)-Glu(5)-His(6)-Phe(7)-Arg(8)-Trp(9)-Gly(10)-Lys(11)-Pro(12)-Val(13)-NH(2), is an endogenous agonist for the melanocortin receptor 1 (MC1R), the receptor found in the skin, several types of immune cells, and other peripheral sites. Three-dimensional models of complexes of this receptor with alphaMSH and its synthetic analog NDP-alphaMSH, Ac-Ser(1)-Tyr(2)-Ser(3)-Nle(4)-Glu(5)-His(6)-D-Phe(7)-Arg(8)-Trp(9)-Gly(10)-Lys(11)-Pro(12)-Val(13)-NH(2), have been previously proposed. In those models, the 6-9 segment of the ligand was considered essential for the ligand-receptor interactions. In this study, we probed the role of Trp(9) of NDP-alphaMSH in interactions with hMC1bR. Analogs of NDP-alphaMSH with various amino acids in place of Trp(9) were synthesized and tested in vitro in receptor affinity binding and cAMP functional assays at human melanocortin receptors 1b, 3, 4, and 5 (hMC1b,3-5R). Several new compounds displayed high agonist potency at hMC1bR (EC(50) = 0.5-5 nM) and receptor subtype selectivity greater than 2000-fold versus hMC3-5R. The Trp(9) residue of NDP-alphaMSH was determined to be not essential for molecular recognition at hMC1bR.     

On the transferability of atomic solvation parameters: Ab initio structural prediction of cyclic heptapeptides in DMSO

A statistical mechanics methodology for predicting the solution structures and populations of peptides developed recently is based on a novel method for optimizing implicit solvation models, which was applied initially to a cyclic hexapeptide in DMSO (C. Baysal and H. Meirovitch, Journal of American Chemical Society, 1998, vol. 120, pp. 800-812). Thus, the molecule has been described by the simplified energy function E(tot) = E(GRO) + summation operator(k) sigma(k)A(k), where E(GRO) is the GROMOS force-field energy, sigma(k) and A(k) are the atomic solvation parameter (ASP) and the solvent accessible surface area of atom k, respectively. In a more recent study, these ASPs have been found to be transferable to the cyclic pentapeptide cyclo(D-Pro(1)-Ala(2)-Ala(3)-Ala(4)-Ala(5)) in DMSO (C. Baysal and H. Meirovitch, Biopolymers, 2000, vol. 53, pp. 423-433). In the present paper, our methodology is applied to the cyclic heptapeptides axinastatin 2 [cyclo(Asn(1)-Pro(2)-Phe(3)-Val(4)-Leu(5)-Pro(6)-Val(7))] and axinastatin 3 [cyclo(Asn(1)-Pro(2)-Phe(3)-Ile(4)-Leu(5)-Pro(6)-Val(7))], in DMSO, which were studied by nmr by Mechnich et al. (Helvetica Chimica Acta, 1997, vol. 80, pp. 1338-1354). The calculations for axinastatin 2 show that special ASPs should be optimized for the partially charged side-chain atoms of Asn while the rest of the atoms take their values derived in our previous work; this suggests that similar optimization might be needed for other side chains as well. The solution structures of these peptides are obtained ab initio (i.e., without using experimental restraints) by an extensive conformational search based on E(GRO) alone and E(*)(tot), which consists of the new set of ASPs. For E(*)(tot), the theoretical values of proton-proton distances, (3)J coupling constants, and other properties are found to agree very well with the nmr results, and they are always better than those based on E(GRO).     

Probing the conformational and dynamical effects of O-glycosylation within the immunodominant region of a MUC1 peptide tumor antigen.

MUC1 mucin is a large transmembrane glycoprotein, the extracellular domain of which is formed by a repeating 20 amino acid sequence, GVTSAPDTRPAPGSTAPPAH. In normal breast epithelial cells, the extracellular domain is densely covered with highly branched complex carbohydrate structures. However, in neoplastic breast tissue, the extracellular domain is under-glycosylated, resulting in the exposure of a highly immunogenic core peptide epitope (PDTRP in bold above), as well as in the exposure of normally cryptic core Tn (GalNAc), STn (sialyl alpha2-6 GalNAc) and TF (Gal beta1-3 GalNAc) carbohydrates. Here, we report the results of 1H NMR structural studies, natural abundance 13C NMR relaxation measurements and distance-restrained MD simulations designed to probe the structural and dynamical effects of Tn-glycosylation within the PDTRP core peptide epitope. Two synthetic peptides were studied: a nine-residue MUC1 peptide of the sequence, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6-Arg7-Pro8-Ala9, and a Tn-glycosylated version of this peptide, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6(alphaGalNAc)-Arg7-Pro8-Ala9. The results of these studies show that a type I beta-turn conformation is adopted by residues PDTR within the PDTRP region of the unglycosylated MUC1 sequence. The existence of a similar beta-turn within the PDTRP core peptide epitope of the under-glycosylated cancer-associated MUC1 mucin protein might explain the immunodominance of this region in vivo, as the presence of defined secondary structure within peptide epitope regions has been correlated with increased immunogenicity in other systems. Our results have also shown that Tn glycosylation at the central threonine within the PDTRP core epitope region shifts the conformational equilibrium away from the type I beta-turn conformation and toward a more rigid and extended state. The significance of these results are discussed in relation to the possible roles that peptide epitope secondary structure and glycosylation state may play in MUC1 tumor immunogenicity.     

Restricted backbone conformational and motional flexibilities of loops containing peptidyl-proline bonds dominate the enzyme activity of staphylococcal nuclease

The role of cis-trans isomerizations of peptidyl-proline bonds in the enzyme activity of staphylococcal nuclease (SNase) was examined by mutation of proline residues. The proline-free SNase ([Pro-]SNase), namely, P11A/P31A/P42A/P47T/P56A/P117G-mutant SNase, was adopted for elucidating the correlation between the nuclease activity and the backbone conformational and dynamic states of SNase. The 3D solution structure of [Pro-]SNase has been determined by heteronuclear NMR experiments. Comparing the structure of [Pro-]SNase with the structure of SNase revealed the conformational differences between the two proteins. In the structure of [Pro-]SNase, conformational rearrangements were observed for the loop of residues Ala112-His121 containing a trans Lys116-Gly117 peptide bond and for the C-terminal alpha-helical loop of residues Leu137-Glu142. Mutation of proline at position 117 also caused the conformational rearrangement of the p-loop (Asp77-Leu89), which is remote from the Ala112-His121 loop. The Ala112-His121 loop and p-loop are placed closer to each other in [Pro-]SNase than in SNase. The backbone dynamic features of the omega-loop (Pro42-Pro56) of SNase are different from those of [Pro-]SNase. The backbone of the omega-loop exhibits restricted flexibility with slow conformational exchange motions in SNase, but is highly flexible in [Pro-]SNase. The analysis indicates that the restrained backbone conformation of the Ala112-His121 loop and restricted flexibility of the omega-loop are two dominant factors determining the enzyme activity of SNase. Of the two factors, the former is correlated with the strained cis Lys116-Pro117 peptide bond and the latter is correlated with the cis-trans isomerizations of the His46-Pro47 peptide bond.     

Phosphorylation causes a conformational change in the carboxyl-terminal domain of the mouse RNA polymerase II largest subunit

The carboxyl-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II can be phosphorylated by a p34cdc2/CDC28-containing CTD kinase. Phosphorylated serine (or threonine) is located at positions 2 and 5 in the repetitive heptapeptide consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. We show here that phosphorylation of the mouse CTD retards its electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels in a way similar to that observed for the II0 form of the largest subunit of RNA polymerase II phosphorylated in vivo. At the maximum level of phosphorylation by CTD kinase in vitro, there are 15-20 phosphates evenly distributed among the 52 heptapeptide repeats that comprise the mouse CTD. Gel filtration chromatography and sucrose gradient ultracentrifugation analyses indicate that phosphorylation induces a dramatic conformational change in the CTD with the phosphorylated form adopting a far more extended structure than the unphosphorylated CTD.     

Empirical solvation models in the context of conformational energy searches: application to bovine pancreatic trypsin inhibitor.

Continuum solvation models that estimate free energies of solvation as a function of solvent accessible surface area are computationally simple enough to be useful for predicting protein conformation. The behavior of three such solvation models has been examined by applying them to the minimization of the conformational energy of bovine pancreatic trypsin inhibitor. The models differ only with regard to how the constants of proportionality between free energy and surface area were derived. Each model was derived by fitting to experimentally measured equilibrium solution properties. For two models, the solution property was free energy of hydration. For the third, the property was NMR coupling constants. The purpose of this study is to determine the effect of applying these solvation models to the nonequilibrium conformations of a protein arising in the course of global searches for conformational energy minima. Two approaches were used: (1) local energy minimization of an ensemble of conformations similar to the equilibrium conformation and (2) global search trajectories using Monte Carlo plus minimization starting from a single conformation similar to the equilibrium conformation. For the two models derived from free energy measurements, it was found that both the global searches and local minimizations yielded conformations more similar to the X-ray crystallographic structures than did searches or local minimizations carried out in the absence of a solvation component of the conformational energy. The model derived from NMR coupling constants behaved similarly to the other models in the context of a global search trajectory. For one of the models derived from measured free energies of hydration, it was found that minimization of an ensemble of near-equilibrium conformations yielded a new ensemble in which the conformation most similar to the X-ray determined structure PTI4 had the lowest total free energy. Despite the simplicity of the continuum solvation models, the final conformation generated in the trajectories for each of the models exhibited some of the characteristics that have been reported for conformations obtained from molecular dynamics simulations in the presence of a bath of explicit water molecules. They have smaller root mean square (rms) deviations from the experimentally determined conformation, fewer incorrect hydrogen bonds, and slightly larger radii of gyration than do conformations derived from search trajectories carried out in the absence of solvent.     

Predicting the conformational states of cyclic tetrapeptides

Biologically active cyclic tetrapeptides, usually found among fungi metabolites, exhibit phytotoxic or cytostatic activities that are likely to be governed by specific conformations adopted in solution. For conformational studies and drug design, there is a strong interest in using fast and reliable methods to determine correctly the conformational population of cyclotetrapeptides. We show here that standard molecular mechanics computational approach gives satisfactory results. The method was validated step by step by experimental data either obtained after synthesis and NMR analysis, or found in the literature. The cyclo(Gly)(4), cyclo(Ala)(4), cyclo(Sar)(4), and cyclo(SarGly)(2) peptides were used to evaluate the prediction of the peptide backbone conformation, and the detailed conformational analysis of tentoxin, a natural phytotoxic cyclotetrapeptide in which N-alkylated peptide bonds alternate with regular secondary ones, was used to validate the computation of conformers proportions. From the knowledge of an initial cyclic primary structure and of the D or L configuration of the amino acids, we show that it is possible to determine the exact orientation of carbonyl groups and to predict the nature of conformers present in solution. The proportion of each conformer can be inferred from a statistical thermodynamics approach by using the potential energy values of each conformer, computed by molecular mechanics methods with the TRIPOS force field, which allowed us to account for the solvent. The solvent contribution was processed by two different methods according to the nature of the interactions: whether through the dielectric constant introduced in the electrostatic potential, when interaction with solute molecules are weak or negligible, or through the computation of free energy of solvation using the algorithm SILVERWARE for solvents explicitly interacting with the solute. When applied to tentoxin, this conformational analysis yielded results in very good agreement with the experimental data reported by Pinet et al. (Biopolymers, 1995, Vol. 36, pp. 135-152), on both the nature of existing conformers and their relative proportions, whatever the nature of the considered solvent.     

Substance P analogs containing alpha,alpha-dialkylated amino acids with potent anticancer activity.

Six analogs (peptides 1-6) of the potent substance P (SP) derivative known as 'Antagonist D' were synthesized by substituting constrained amino acids Aib or Acp (cycloleucine, 1-amino cyclopentane carboxylic acid) at different positions in the Antagonist D sequence: D-Arg(1)-Pro(2)-Lys(3)-Pro(4)-D-Phe(5)-Gln(6)-D-Trp(7)-Phe(8)-D-Trp(9)-Leu(10)-Leu(11)-NH(2). In the preliminary in vitro antiproliferative screening of the analogs on different human cancer cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, peptide 1 was found to be the most active. Further, peptide 1 was butanoylated (analog 5) or octanoylated (analog 6) at the N-terminus. SP analogs 1, 5, and 6 were evaluated in vivo in a xenograft model of human primary colon tumor (PTC) cell line in athymic nude mice and were found to cause tumor regression. This study investigates if the use of the constrained amino acids Aib and Acp in the designed SP analogs can retain the in vitro and in vivo anticancer activities, which could be useful in cancer therapy and drug targeting. Further, the strategy of incorporation of Aib or Acp in biologically active peptides can be exploited in determining the receptor-bound conformation and in transforming these bioactive peptides into pharmacologically useful drugs.