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.
Uchiki
Tomoaki
T
Genome Science and Technology Graduate School, University of Tennessee-Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA.
Hettich
Robert
R
Gupta
Vibha
V
Dealwis
Chris
C
D000595:Amino Acid Sequence; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology; D005656:Fungal Proteins / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D015003:Yeasts
D004220:Disulfides; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; C115052:SML1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012264:Ribonucleotide Reductases