Examining protein surface structure in highly conserved sequence variants with mass spectrometry.
A simple mass spectrometry-based method capable of examining protein structure called SNAPP (selective noncovalent adduct protein probing) is used to evaluate the structural consequences of point mutations in naturally occurring sequence variants from different species. SNAPP monitors changes in the attachment of noncovalent adducts to proteins as a function of structural state. Mutations that lead to perturbations to the electrostatic surface structure of a protein affect noncovalent attachment and are easily observed with SNAPP. Mutations that do not alter the tertiary structure or electrostatic surface structure yield similar results by SNAPP. For example, bovine, porcine, and human insulin all have very similar backbone structures and no basic or acidic residue mutations, and the SNAPP distributions for all three proteins are very similar. In contrast, four variants of cytochrome c (cytc) have varying degrees of sequence homology, which are reflected in the observed SNAPP distributions. Bovine and pigeon cytc have several basic or acidic residue substitutions relative to horse cytc, but the SNAPP distributions for all three proteins are similar. This suggests that these mutations do not significantly influence the protein surface structure. On the other hand, yeast cytc has the least sequence homology and exhibits a unique, though related, SNAPP distribution. Even greater differences are observed for lysozyme. Hen and human lysozyme have identical tertiary structures but significant variations in the locations of numerous basic and acidic residues. The SNAPP distributions are quite distinct for the two forms of lysozyme, suggesting significant differences in the surface structures. In summary, SNAPP experiments are relatively easy to perform, require minimal sample consumption, and provide a facile route for comparison of protein surface structure between highly homologous proteins.
Department of Chemistry, University of California, Riverside, California 92521, United States.
Yuanqi
Y
Tao
Ryan R
RR
Julian
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002645:Chickens; D010856:Columbidae; D017124:Conserved Sequence; D045304:Cytochromes c / Q000737:chemistry; D006736:Horses; D006801:Humans; D007328:Insulin / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D013552:Swine; D015003:Yeasts
D007328:Insulin; D011506:Proteins; D045304:Cytochromes c; D009113:Muramidase