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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.

Sml1p is small protein that binds to and inhibits the activity of ribonucleotide reductase (RNR) 3 , a protein enzyme complex that controls the balance and level of the cellular deoxynucleotide diphosphate pools that are critical for DNA synthesis and repair. In this respect, Sml1p is a checkpoint protein whose function is to regulate the activity of the large subunit of RNR (Rnr1p). Sml1p is thought to be regulated by the MEC1/RAD53 cell cycle checkpoint pathway. Neither the structure of Sml1p nor its complex to Rnr1p is well known. In this report, we describe how a recombinant Sml1p-histag protein (in both monomeric and dimeric forms) can be characterized with electrospray mass spectrometry. Mass spectrometry can play a vital role in the study of the Sml1p-Rnr1p complex by: ( 1 ) confirming the identities and purities of recombinant proteins such as Sm1lp-histag (with mass accuracy and resolution far superior to SDS–PAGE) and ( 2 ) verifying the presence or absence of PTM, chemical modifications, or metal–ion binding to the protein species, which may alter the function and binding of the protein partners.