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S R Lehrman

Publications and source records attributed to S R Lehrman.

8 recordsLinked to original sources
PubMed1992

Identification and characterization of an anti-isoaspartic acid monoclonal antibody.

The deamidation and rearrangement of protein-bound asparagine residues occurs when peptides and proteins are exposed to acidic or alkaline aqueous media. Asn99 of bovine growth hormone (bGH) is readily modified via these mechanisms. We have generated a monoclonal antibody (MAb) that interacts with a bGH fragment that contains an isoaspartyl residue. To obtain this antibody, CAF1/J mice were immunized with [isoaspartyl99]-bGH(96-112) conjugated to BSA. Using a competitive ELISA assay, the interaction of this MAb to [isoaspartyl99]-bGH(96-112) has been observed to have an apparent Km of 150 nM. The corresponding native peptide and other bGH fragments do not bind to this antibody with high affinity. For example, the binding affinities of [Asp99]-bGH(96-112) and [Glu99]-bGH(96-112) to this antibody are 54- and 78-fold lower than the corresponding isoaspartyl peptide. The antibody also binds to bGH that is enriched in isoaspartic acid at position 99, but not to the unmodified protein. The binding epitope of the peptide has been further characterized by comparing the binding of bGH(96-112) analogues to the MAb. Alanine substitution at residues 99, 100, 101, and 103 reduce binding affinity to the antibody by more than 10(3)-fold. Replacement of valine with alanine at position 102 has much less impact on antibody affinity. Further experiments suggest that the relative insensitivity to this substitution is due to the structural similarity of these sidechains. Other isoaspartic acid-containing peptides not derived from the bGH sequence do not bind to the antibody. We conclude that the epitope binding site of this MAb is highly specific for 99-103 of [isoaspartyl99]-bGH (96-112).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Site-directed mutagenesis to probe protein folding: evidence that the formation and aggregation of a bovine growth hormone folding intermediate are dissociable processes.

Bovine growth hormone (bGH) forms a stable folding intermediate that aggregates at elevated concentrations (greater than 10 microM). Thermodynamic and kinetic studies have shown that the formation of this bGH folding intermediate and its aggregation are separate processes, implying that selective modifications of bGH can lead to their independent modulation. In addition, a bGH region that includes amino acid residues 109-133 appears to be directly involved in this aggregation process. Human growth hormone (hGH), which is unable to aggregate via this mechanism, differs from the bovine primary sequence at eight positions within this protein region. We have characterized the folding of a bGH analogue that contains the hGH sequence between amino acid residues 109-133 (8H-bGH) at low and high concentrations. The equilibrium folding characteristics of bGH and 8H-bGH are similar when monitored at low protein concentrations (less than or equal to 2 microM). The wild-type and analogue proteins have equivalent denaturation midpoints when equilibrium unfolding is monitored by the use of far-UV circular dichroism, second-derivative UV, or fluorescence. In addition, the enhanced fluorescence that is associated with the formation of the bGH monomeric folding intermediate (Havel, H. A., et al. (1988) Biochim. Biophys. Acta 955, 154-163) is observed for 8H-bGH under similar conditions. In contrast, partial denaturation of 8H-bGH at higher concentrations (greater than 2 microM) leads to significantly less aggregation than is observed for bGH. This result is obtained from near-UV CD spectroscopy, kinetic folding, size-exclusion chromatography, and dynamic light-scattering data.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Peptide alpha-helicity in aqueous trifluoroethanol: correlations with predicted alpha-helicity and the secondary structure of the corresponding regions of bovine growth hormone.

The relationship between trifluoroethanol (TFE) enhancement of peptide alpha-helicity and protein secondary structure has been studied for a series of 11 peptides which span the complete primary sequence of bovine growth hormone (bGH). Ten of these peptides become increasingly alpha-helical as the solution concentration of TFE is increased. The amount of alpha-helicity developed by these peptides plateaus above 10 mol % TFE and ranges from 0 to 71%. The increased alpha-helicity, as determined by CD, closely correlates with the amount of alpha-helix predicted for eight of the eleven peptides analyzed (r = 0.9). Therefore, for this group of peptides, it appears that this technique can be used as a measure of alpha-helical propensity. Inclusion of the remaining three peptides in this analysis significantly lowers the correlation (r = 0.6). The reduced correspondence between TFE-enhanced and predicted alpha-helicity in this latter subset of peptides may be due to their relatively high hydrophobicity. In addition, the relevance of TFE-enhanced peptide alpha-helicity and the secondary structure of the corresponding protein regions was explored. Although the three peptides which form the largest amount of alpha-helicity in the presence of 10 mol % TFE correspond to alpha-helical regions of the protein, the overall correlation is significantly lower than is observed for the TFE-enhanced and predicted alpha-helicity. These findings suggest that the propensity of specific amino acid sequences for alpha-helix formation influences the amount of alpha-helicity which forms in corresponding protein sequences, but that other factors can modify this structure.

Amino Acid Sequence↗
PubMed1990

Protein structure.

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Amino Acid Sequence↗
PubMed1988

Primary structure of equine pituitary prolactin.

Equine prolactin was determined to be a single chain protein of 199 amino acid containing two tryptophan and six cysteine residues, as found in other mammalian prolactins. The primary sequence of equine prolactin was obtained by automated Edman analyses of S-carboxymethylated protein and proteolytic fragments of modified protein. Of the known prolactin sequences, equine prolactin shows closest homology with porcine (93%) and fin whale (87-91%) prolactins. Genetic mutations have produced changes in 17 of 199 residues of equine prolactin relative to its putative ancestral precursor. Since equine growth hormone has undergone alterations in 4 of 191 residues relative to this putative precursor protein, these results support the theory that prolactins are evolving at a faster rate than growth hormones. Consistent with the previously determined circular dichroic spectrum of equine prolactin, 60% of the protein is predicted to form alpha helices.

Amino Acid Sequence↗
PubMed1987

Helical formation in isolated fragments of bovine growth hormone.

The peptide 109-133 was isolated from bovine growth hormone (bGH) and studied for helix formation in aqueous solutions. This fragment was shown to contain helical structure by far-ultraviolet circular dichroism in aqueous solutions. The amount of helix was dependent on pH and peptide concentration. The peptide has maximum helicity between pH 4 and 5 and at high peptide concentration. Under these conditions for maximal helix population, this fragment is approximately 100% helical. Secondary structure predictions suggest that residues 110-127 have a strong propensity to form an amphipathic helix. We have also studied a related peptide, 96-133, and show by gel filtration that it undergoes an increase in molecular weight that directly correlates with a coil to helix transition. A comparison of the helical content of 96-133 to 109-133 and circular dichroism studies of peptide 96-112 suggest that the helix of 96-133 is limited to the 109-133 region. Current models for alpha-helix formation predict that peptides the size of 109-133 should not contain measurable helicity in aqueous solutions. Our studies show that the unusual stability of helix 109-133 is due to electrostatic interactions and probable intermolecular packing between hydrophobic faces of the amphipathic surfaces of the helices. The implications of helix formation in these fragments to a framework model of protein folding for bGH are discussed.

Amino Acid Sequence↗