Stabilization of a type VI turn in a family of linear peptides in water solution.

The sources of the stability of a type VI turn formed with high population in the cis isomeric form of an unblocked six residue peptide, Ser1-Tyr2-Pro3-Tyr4-Asp5-Val6 (SYPYDV), were investigated by making extensive amino acid substitutions at residues 2, 4 and 5. Several NMR parameters indicate the presence of the turn, including significant upfield shifts of the proton resonances of the cis proline, a small 3JHN alpha coupling constant for residue 2, a cross-turn d alpha N(i,i+2) from residue 2 to residue 4 and in increased mole fraction of the cis form in the conformational ensemble. By these criteria, a number of peptides were found to contain significant populations of type VI turn conformers in the cis form of the peptide. The NMR parameters are highly dependent on the sequence of the peptide, and are strongly correlated with each other and with the population of type VI turn. The greatest populations of turn conformations were observed for peptides of the general form AA-Ar-Pro-Ar-Hp, where AA represents any amino acid, Ar an aromatic residue and Hp a small hydrophilic residue. There is no evidence in the form of lowered amide proton temperature coefficients for direct hydrogen bonding as a primary source of turn stability. Instead, the major stabilizing factor, indicated by the strong dependence of the turn population on the presence of aromatic (not hydrophobic) residues at positions 2 and 4, is the stacking of the aromatic and proline rings. A measurable preference for deprotonated aspartate at position 5, which is not part of the turn itself, and the destabilization of the turn at high and low pH, indicate that electrostatic interactions between the unblocked N terminus and the aspartate carboxyl group also act to stabilize the turn conformation when the Ar-Pro-Ar sequence is present. Implications for stabilization of local elements of secondary structure during the earliest events in protein folding are discussed.

Folding propensities of peptide fragments of myoglobin.

Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthetized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformation preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the alpha region of (phi, psi) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A- and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate beta-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, either studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding.

Truncated, branched, and/or cyclic analogues of neuropeptide Y: importance of the pancreatic peptide fold in the design of specific Y2 receptor ligands.

Truncated, branched, and/or cyclic neuropeptide Y (NPY) analogues were tested for their ability to bind to the neuroblastoma cells, SK-N-MC (Y1 receptor) and SK-N-BE(2) (Y2 receptor). The design of such analogues was inspired by models of NPY based on the crystal structure of avian pancreatic polypeptide. The minimum length of the backbone was investigated using the following truncated analogues [binding affinity (nM) for Y1 and Y2 receptor subtypes respectively are given in parentheses]: des-AA10-17[D-Ala9]NPY (100, 0.9), des-AA7-23[D-Ala6]NPY (> 1000, 1.2), des-AA4-26[D-Ala3]NPY (> 1000, 120), cyclo(7,20)-des-AA10-17[Glu7,D- Ala9,D-Dpr20]NPY (100, nd), cyclo(2,27)-des-AA7-23[Glu2,D-Ala6,D-Dpr27]NPY (> 1000, 3.6), cyclo(2,30)- des-AA7-23[Glu2,D-Ala6,-D-Dpr30]NPY (> 1000, nd), cyclo(1,30)-des-AA4-26[Glu1,D-Ala3,D-Dpr30]NPY (> 1000, > 1000). A new family of branched NPY analogues corresponding to the partial deletion of the polyproline helix with conservation of the N-terminus was also examined: des-AA7-23[(Ac-NPY14-22)-epsilon-D-Lys6]NPY (> 1000, 2.1), des-AA7-23[(Ac-NPY7-22)-epsilon-D-Lys6]NPY (> 1000, 5.1), des-AA7-23-[(Ac-LEALEG-NPY14-22)-epsilon-D-Lys6]NPY (> 1000, 4.8). Finally, the role played by the flexible tail (residues 32-36) was studied with the following cyclic analogues: cyclo(30,34)-[Lys30,Glu34]NPY18-36 (> 1000, 360), cyclo(30,34)-[Orn30,Gly34]NPY18-36 (> 1000, 950), cyclo(30,34)-[Dpr30,Glu34]NPY18-36 (> 1000, 590), cyclo(33,36)-[Lys33,Glu36]NPY (> 1000, > 1000), cyclo(33,36)-[Lys33,Glu36]NPY18-36 (> 1000, > 1000). These results suggest that the Y1 receptor is highly discriminatory since deletion of residues 10-17, shown to have little effect on Y2 binding affinity, reduces Y1 affinity 50-fold. Bridging sites and constructs have been identified that may serve as useful leads in the design of more potent and selective analogues. We have identified two positions (9 and 6) where the introduction of a D amino acid is not detrimental to binding affinity. Whether this modification leads to the stabilization of a yet unidentified turn compatible with high Y2 receptor affinity will have to be determined by spectroscopic methods. Finally, stabilizing a putative alpha-helical conformation of the C-terminal heptapeptide of NPY18-36 has a deleterious effect on the Y1 and Y2 receptors.

Pivalase catalytic antibodies: towards abzymatic activation of prodrugs.

Screening of monoclonal-antibody libraries generated against the tert-butyl phosphonate hapten 2 and the chloromethyl phosphonate hapten 3 with pivaloyloxymethyl-umbelliferone 1 as a fluorogenic substrate led to the isolation of eleven catalytic antibodies with rate accelerations around kcat/ kuncat = 10(3). The antibodies are not inhibited by the product and accept different acyloxymethyl derivatives of acidic phenols as substrates. The highest activity was found for the bulky, chemically less-reactive pivaloyloxymethyl group: there is no activity with acetoxymethyl or acetyl esters. This difference might reflect the preference of the immune system for hydrophobic interactions in binding and catalysis. Pivalase catalytic antibodies might be useful for activating orally available pivaloyloxymethyl prodrugs.

NMR characterization of a single-cysteine mutant of Escherichia coli thioredoxin and a covalent thioredoxin-peptide complex.

The mechanism of disulfide reduction by thioredoxin in the cell is thought to occur through the formation and subsequent destruction of a mixed-disulfide intermediate between thioredoxin and the substrate. In order to model the interaction, we have prepared a mutant of Escherichia coli thioredoxin where the second cysteine residue of the active site has been replaced by an alanine residue. A specific covalent complex has been prepared between the remaining cysteine residue and a short cysteine-containing peptide. This paper describes the preparation and characterization of the mutant protein both free and in the peptide complex.

Contribution of increased length and intact capping sequences to the conformational preference for helix in a 31-residue peptide from the C terminus of myohemerythrin.

In order to examine the effects of chain length on the propensity of short peptides to form helix-like structures in aqueous solution, we have studied a peptide of 31 residues consisting of the C-terminal sequence (residues 88-118) of the four-helix bundle protein myohemerythrin from Themiste zostericola. This peptide, termed MDC, represents the final two elements of secondary structure in the protein, the D-helix and the C-terminal loop sequence, together with a five-residue sequence at the N terminus corresponding to the linker between the C- and D-helices. An N-capping sequence, VDAKNV, immediately precedes the D-helix sequence, and a C-capping sequence, VNHIKGT, corresponding to the alphaL termination motif, occurs at the C-terminal end. The effect of replacement of a cysteine residue in the middle of the sequence with an alanine was explored by the comparison of the MDC peptide and a 16-residue peptide representing the sequence of the D-helix alone, both containing the change Cys99Ala. Significant changes in the NMR and CD spectra were seen for both peptides compared to the wild-type sequence. A comparison of the fluorescence spectra of the wild-type and Cys99Ala peptides indicated that a specific interaction between the side chains of Cys 99 and Trp 102 acts to quench the fluorescence of the tryptophan ring and probably contributes a component that distorts the CD spectrum of the wild-type peptide at approximately 220-235 nm. The effect of an increase in the length of the peptide, with the incorporation of capping sequences derived from the native sequence, was explored by NMR and CD spectroscopy of the 31-residue and 16-residue peptides in aqueous solution and in TFE/water mixtures. Evidence for the formation of a significant population of helical conformers in the region of the MDC peptide corresponding to the D-helix was observed in aqueous solution using CD and NMR spectroscopy. The C-terminal 10 residues of the MDC peptide behave in solution in a manner identical to that of a 10-residue peptide with the same sequence; a highly specific local interaction between an aromatic ring and a glycine amide proton appears to be retained in the longer peptide. Upon addition of trifluoroethanol (TFE), significant shifts are observed in a number of resonances in the NMR spectrum, and both chemical shifts and NOEs provide evidence for a higher population of helix in the D-helix region of the peptide in TFE. However, TFE is unable to promote the propagation of helix beyond the N-cap or alphaL termination motifs, and the specific local interaction observed in the C-terminal sequence is retained in TFE. The CD spectrum in TFE shows an increase in the proportion of helix, to an overall maximum of approximately 55% helix at 50% v/v TFE, corresponding to approximately 100% helix in the D-helix sequence of the peptide, since the N and C termini of the MDC peptide are not helical according to the NMR spectra. The high proportion of helix observed in the D-helix sequence of the longer MDC peptide demonstrates that the presence of intact capping sequences can constrain the peptide conformational ensemble to resemble that seen in the native protein. A compendium of results from this and previous peptide studies has also led to a novel observation, the existence of a correlation between the amide proton chemical shift and temperature coefficient.