Publications

Hydroxyl radical surface mapping is a useful tool for investigating protein structure and folding. The rate of protein side-chain oxidation by the hydroxyl radical is known to be affected primarily by the chemical reactivity of the side chain and the accessibility of the reactive site to the radical. Efforts have been made to determine the inherent rate of stable product formation of each amino acid side chain, so that the rate of oxidation of an amino acid can be used to accurately estimate the average solvent accessibility of the amino acid side chain in the folded protein. However, the effects of nearby primary sequence on peptide oxidation have not been studied. Here, we examine the amounts of various oxidation products of a small peptide consisting of one leucine and one aspartic acid separated by zero to five glycine residues, as well as with modification of the N- and C-terminus. We find that the relative amounts of certain oxidation products can be heavily influenced by the primary structure of the surrounding peptide. The formation of many products, including hydroxylation, is inhibited by proximity to negative charges, while the formation of other products showed more complicated responses to changing primary sequence.

Derivatization of a variety of peptides by a method known to enhance anhydride formation is demonstrated by mass spectrometry to yield ions that have elemental composition and fragmentation properties identical to [b(n-1) + OH + H]+ ions formed by gas-phase rearrangement and fragmentation. The [b(n-1) + OH + H]+ ions formed by gas-phase rearrangement and fragmentation and the solution-phase [b(n-1) + OH + H]+ ion structural analogs formed by derivatization chemistry show two different forms of dissociation using multiple-collision CAD in a quadrupole ion trap and unimolecular decomposition in a TOF-TOF; one group yields identical product ions as a truncated form of the peptide with a free C-terminal carboxylic acid and fragments at the same activation energy; the other group fragments differently from the truncated peptide, being more resistant to fragmentation than the truncated peptide and yielding primarily the [b(n-2) + OH + H]+ product ion. Nonergodic electron capture dissociation MS/MS suggests that any structural differences between the specific-fragmenting [b(n-1) + OH + H]+ ions and the truncated peptide is at the C-terminus of the peptide. The specific-fragmentation can be readily observed by MS(n) experiments to occur in an iterative fashion, suggesting that the C-terminal structure of the original [b(n-1) + OH + H]+ ion is maintained after subsequent rearrangement and fragmentation events in peptides which fragment specifically. A mechanism for the formation of specific-fragmenting and nonspecific-fragmenting [b(n-1) + OH + H]+ ions is proposed.

Photochemically generated hydroxyl radicals were used to map solvent-exposed regions in the C14S mutant of the protein Sml1p, a regulator of the ribonuclease reductase enzyme Rnr1p in Saccharomyces cerevisiae. By using high-performance mass spectrometry to characterize the oxidized peptides created by the hydroxyl radical reactions, amino acid solvent-accessibility data for native and denatured C14S Sml1p that revealed a solvent-excluding tertiary structure in the native state were obtained. The data on solvent accessibilities of various amino acids within the protein were then utilized to evaluate the de novo computational models generated by the HMMSTR/Rosetta server. The top five models initially generated by the server all disagreed with both published nuclear magnetic resonance (NMR) data and the solvent-accessibility data obtained in this study. A structural model adjusted to fit the previously reported NMR data satisfied most of the solvent-accessibility constraints. Through minor adjustment of the rotamers of two amino acid side chains for this latter structure, a model that not only provided a lower energy conformation but also completely satisfied previously reported data from NMR and tryptophan fluorescence measurements, in addition to the solvent-accessibility data presented here, was generated.

Protein surfaces are important in most biological processes, including protein-protein interactions, enzymatic catalysis, and protein-ligand binding. We report a method in which hydroxyl radicals generated by a rapid-UV irradiation of a 15% hydrogen peroxide solution were utilized to oxidize specific amino acid side chains of two model proteins (lysozyme, beta-lactoglobulin A), according to the residues' chemical reactivities and the solvent accessibility of the reactive carbons and sulfurs in the residue. Oxidized peptides generated by tryptic digestion were identified by electrospray-Fourier transform mass spectrometry. The specific sites of the stable modification were then identified by reverse-phase liquid chromatography coupled to quadropole ion trap tandem mass spectrometry. The solvent accessibility of the residue was shown to directly affect the rate of oxidation by this method (with the exception of methionine), supporting its use as a rapid measure of the solvent accessibility of specific residues, and in some cases, individual atoms.

The solvent-accessible surface area of proteins is important in biological function for many reasons, including protein-protein interactions, protein folding, and catalytic sites. Here we present a chemical technique to oxidize amino acid side chains in a model protein, apomyoglobin, and subsequent elucidation of the effect of solvent accessibility on the sites of oxidation. Under conditions of low protein oxidation (zero to three oxygen atoms added per apomyoglobin molecule), we have positively identified five oxidation sites by liquid chromatography-tandem mass spectrometry and high-resolution Fourier transform mass spectrometry. Our results indicate that all oxidized amino acids, with the exception of methionine, have highly solvent-accessible side chains, but the rate of oxidation may not be dictated solely by solvent accessibility and amino acid identity.

We have identified nine oligopeptide transporter (OPT) orthologs (AtOPT1 to AtOPT9) in Arabidopsis. These proteins show significant sequence similarity to OPTs of Candida albicans (CaOpt1p), Schizosaccharomyces pombe (Isp4p), and Saccharomyces cerevisiae (Opt1p and Opt2p). Hydrophilicity plots of the OPTs suggest that they are integral membrane proteins with 12 to 14 transmembrane domains. Sequence comparisons showed that the AtOPTs form a distinct subfamily when compared with the fungal OPTs. Two highly conserved motifs (NPG and KIPPR) were found among all OPT members. The identification of multiple OPTs in Arabidopsis suggests that they may play different functional roles. This idea is supported by the fact that AtOPTs have a distinct, tissue-specific expression pattern. The cDNAs encoding seven of the AtOPTs were cloned into a yeast vector under the control of a constitutive promoter. AtOPT4 expressed in S. cerevisiae mediated the uptake of KLG-[3H]L. Similarly, expression of five of the seven AtOPT proteins expressed in yeast conferred the ability to uptake tetra- and pentapeptides as measured by growth. This study provides new evidence for multiple peptide transporter systems in Arabidopsis, suggesting an important physiological role for small peptides in plants.