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 synthesized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformational 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 a region of (c#J,$) 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 p-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, earlier 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.Keywords: myoglobin; nuclear magnetic resonance; peptide conformation; protein folding; reverse turn; secondary structureThe mechanism by which proteins fold into their native conformations remains one of the central unsolved problems in molecular biology. One view is that folding occurs along defined pathways, often with structured intermediates (for reviews, see Creighton,
We report the NMR resonance assignments for a macromolecular protein/DNA complex containing the three amino-terminal zinc fingers (92 amino acid residues) of Xenopus laevis TFIIIA (termed zf1-3) bound to the physiological DNA target (15 base pairs), and for the free DNA. Comparisons are made of the chemical shifts of protein backbone 1HN, 15N, 13C alpha and 13C beta and DNA base and sugar protons of the free and bound species. Chemical shift changes are analyzed in the context of the structures of the zf1-3/DNA complex to assess the utility of chemical shift change as a probe of molecular interfaces. Chemical shift perturbations that occur upon binding in the zf1-3/DNA complex do not correspond directly to the structural interface, but rather arise from a number of direct and indirect structural and dynamic effects.
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 presen...
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.
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