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

Genome Science and Technology Graduate School, University of Tennessee-Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA.
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.
Mesh Terms:
Amino Acid Sequence, Dimerization, Disulfides, Enzyme Inhibitors, Fungal Proteins, Macromolecular Substances, Molecular Sequence Data, Molecular Weight, Recombinant Proteins, Ribonucleotide Reductases, Saccharomyces cerevisiae Proteins, Spectrometry, Mass, Electrospray Ionization, Yeasts
Anal. Biochem. Feb. 01, 2002; 301(1);35-48 [PUBMED:11811965]
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