Characterization of monomeric and dimeric forms of recombinant Sml1p-histag protein by electrospray mass spectrometry.

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.