Biomedical subjects

P D Semchuk

Publications and source records attributed to P D Semchuk.

6 recordsLinked to original sources

PubMed1999

Hydrophilic interaction/cation-exchange chromatography for the purification of synthetic peptides from closely related impurities: serine side-chain acetylated peptides.

Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEC) is a novel HPLC technique which has excellent potential for peptide separations. Separations by HILIC/CEC are carried out by subjecting peptides to linear increasing salt gradients in the presence of high levels of acetonitrile, which promotes hydrophilic interactions overlayed on ionic interactions with the cation-exchange matrix. Complex peptide mixtures produced by solid-phase synthesis are a frequently encountered and challenging purification problem. In the present study a two-step protocol, consisting of HILIC/CEC followed by RPC, was required for the successful purification of a 21-residue synthetic amphipathic alpha-helical peptide from serine side-chain acetylated impurities, with HILIC/CEC proving to be highly sensitive to subtle differences in hydrophilicities between the acetylated peptides and the desired product. Investigation of the three potential sites of serine acetylation through solid-phase synthesis of acetylated analogues of the desired peptide (peptides of the same sequence and secondary structure, but acetylated at different positions on the hydrophilic face of the alpha-helix) demonstrated that acetylation was occurring at different sites on the peptide. HILIC/CEC was able to take advantage of very subtle changes in environment around the acetylation sites and thus effect a separation of these analogues not achievable by RPC or CEC alone.

Acetylation↗

PubMed1998

Liquid chromatographic-high-resolution mass spectrometric and tandem mass spectrometric identification of synthetic peptides using electrospray ionization.

Liquid chromatography-high-resolution electrospray mass spectrometry (LC-ESI-MS) was investigated for the identification of known and unknown synthetic peptides in a research effort designed to evaluate the applicability of this and complementary MS techniques for peptide characterization and identification. The monoisotopic molecular masses of five related peptides with molecular masses between 2000 and 2500 u were acquired with a resolution of 3000 (10% valley). Under narrow and wide mass range magnetic sector scanning conditions monoisotopic molecular mass errors were typically in the 10-20 and 30-40 ppm range, respectively. Tryptic maps were generated for each peptide following LC-ESI-MS analysis and collisionally activated dissociation (CAD) in the ESI interface resulted in the production of characteristic product ions that enabled amino acid sequencing of the tryptic fragments. Unknown identification was demonstrated during analysis of an incomplete synthetic peptide reaction mixture. The synthesis of an 18 amino acid peptide, LTTAVKKVLTTGLPALIS, was not successful. In its place were six unknown peptides that were identified on the basis of monoisotopic molecular mass and amino acid sequence data. The monoisotopic molecular masses of these unknowns were determined to within 10-20 ppm with a resolution of 3500 (10% valley). Amino acid sequences for the six peptides were generated during ESI-MS-MS analysis. Finally two synthetic peptides differing only by the incorporation of a 13C at leucine were analysed with a resolution of 6000 (10% valley) to confirm that the isotopic distributions were consistent with theoretical expectations.

Amino Acid Sequence↗

PubMed1995

High resolution electrospray mass spectrometry with a magnetic sector instrument: accurate mass measurement and peptide sequencing.

The accurate molecular weights for a series of 37 unknown synthetic peptides, used in research studies involving synthetic vaccines, antibacterial peptides or the de novo design of helical peptides and proteins, were determined with a magnetic sector instrument. All data were obtained with external calibration over a wide mass range during magnetic scanning. Errors between observed and theoretical monoisotopic molecular weights were typically in the 5-60 ppm range for the unknowns at sector resolutions between 2500 and 9000 (10% valley). Isotopic clusters for charge states up to 10+ were resolved through the use of high resolution. Collisionally activated dissociation (CAD) in the electrospray interface resulted in product ions that enabled either full or partial sequencing of most unknown peptides of molecular weights below 2000 Da. The complete primary sequence for one peptide was determined and the importance of high resolution was demonstrated by the differentiation of lysine from glutamine, two amino acids differing in residue mass by only 0.0364 Da. Two other peptides, with identical monoisotopic masses, but different primary sequences, were differentiated based on CAD-MS data.

Amino Acid Sequence↗

PubMedDate not supplied

Design, synthesis and structural characterization of model heterodimeric coiled-coil proteins.

We report the design and synthesis of model heterodimeric coiled-coil proteins and the packing contribution of interchain hetero-hydrophobic side-chains to coiled-coil stability. The heterodimeric coiled-coils are obtained by oxidizing two 35-residue polypeptide chains, each containing a cysteine residue at position 2 and differing in amino acid sequences in the hydrophobic positions ("a" and "d") responsible for the formation and stabilization of the coiled-coil. In each peptide, a single Ala residue was substituted for Leu at position "a" or "d". The formation and stability of heterodimeric coiled-coils were investigated by circular dichroism studies in the presence and absence of guanidine hydrochloride and compared to the corresponding homodimeric coiled-coils. The coiled-coil proteins with an Ala substitution at position "a" were less stable than those with an Ala substitution at position "d" in both the homodimeric (Ala-Ala interchain interactions) and heterodimeric (Leu-Ala interchain interactions ) coiled-coils. The 70-residue disulfide bridged peptides (homo- and heterodimeric coiled-coils) can be readily separated by reversed-phase chromatography (RPC) even though they have identical amino acid compositions as well as in the hydrophobic "a" and "d" positions. The elution of the 70-residue peptides prior to their corresponding 35-residue monomers suggests that these proteins are retaining a large portion of their coiled-coil structure during RPC at pH2 and their retention behavior correlates with protein stability.

Amino Acid Sequence↗

PubMedDate not supplied

Synthetic model proteins: contribution of hydrophobic residues and disulfide bonds to protein stability.

The objective of this study was to evaluate quantitatively the contribution that hydrophobic residues and disulfide bonds make to protein stability using a model protein with well-defined secondary, tertiary and quaternary structure. This de novo-designed protein consists of two identical 35-residue alpha-helical polypeptide chains arranged in a coiled-coil structure, which are stabilized by nine pairs of interchain hydrophobic interactions from leucine residues and an interchain disulfide bond. The mutant proteins differ from the Leu-protein only at positions 16 and 19 of each chain which contain either Ile, Val, Ala, Phe or Tyr instead of Leu residues. The stability of each protein was determined in its reduced and oxidized form from guanidine hydrochloride denaturation experiments using circular dichroism measurements. The Leu-, Ile-, Val- and Phe-proteins containing the disulfide bond were essentially 100% alpha-helical in benign medium (0.05 M phosphate buffer, pH 7, containing 0.1 M KCl) with transition midpoints of 5.3, 4.1, 2.9 and 2.4 M denaturant, respectively. Reduction of the disulfide bond had little effect on the Leu-protein but resulted in significant decreases in helicity of the other mutants. The order of protein stability of these analogs remains the same in the absence or presence of the disulfide bond (Leu- greater than Ile- greater than Val- greater than Phe- greater than Tyr- greater than Ala-protein). Interestingly, the more stable the protein was in the absence of the disulfide bond, the larger the contribution the disulfide bond made to protein stability. The identical mutation in the disulfide bridge protein had a much greater effect on protein stability than in the reduced protein. This suggests that the disulfide bond controls the conformation of the coiled-coil some 14-17 residues further along the polypeptide chain (the disulfide bond is between positions 2 and 2' and the closest mutation site in the coiled-coil is at position 16 and 16'). In contrast, in the absence of the disulfide bond, the coiled-coil is more flexible and can accommodate the mutations more easily by adjusting the interchain packing around the mutation sites. The helix-forming tendency of nonpolar residues was deduced in this study by comparing the alpha-helicity of the mutant polypeptides in aqueous buffer containing 50% trifluoroethanol where they exist as single-stranded helices. Hydrophobicity is an important factor in determining the alpha-helix forming tendency of aliphatic nonpolar residues in amphipathic alpha-helices.

Amino Acid Sequence↗

PubMedDate not supplied

Protein design using model synthetic peptides.

We have designed and synthesized a small, unique protein molecule with defined secondary, tertiary and quaternary structure. This 35-residue peptide, containing a cysteine residue at its N-terminal end, was oxidized to form a 70-residue disulfide-linked two-stranded alpha-helical coiled-coil with the two alpha-helices parallel and in-register. The major contribution to the formation and stabilization of the alpha-helical coiled-coil is hydrophobic interactions between positions 2 and 5 of the heptapeptide repeat (Lys-Leu-Glu-Ala-Leu-Glu-Gly). The protein (L-protein) contains nine leucine-leucine hydrophobic interactions between the alpha-helices of the coiled-coil. Circular dichroism studies demonstrated that this protein in its reduced ([L (r)] or oxidized (L (o)] state was essentially 100% alpha-helical ([theta]220 = -34,050 and -32,000 degrees respectively) at pH 2 (0.1% aqueous trifluoroacetic acid). Our objective was to modify systematically the structure of L to delineate the contribution that various amino acid side chains make to the formation and stabilization of its three-dimensional structure. A-protein, which contains alanine instead of leucine at positions 16 and 19 of the hydrophobic repeat in each chain of the coiled-coil, was compared to the L-protein. At pH 2, the oxidized form of the A-protein [A (o)] was essentially 100% helical. However, the protein was much less stable to temperature denaturation compared to the L-protein. The replacement of two leucine-leucine interactions by two alanine-alanine interactions has a dramatic effect on the formation and stability of the two-stranded alpha-helical coiled-coil structure. The results of this study clearly demonstrate the validity of this synthetic model protein approach to understanding the molecular aspects responsible for the folding and stabilization of protein molecules.

Amino Acid Sequence↗