Characterization of Monomeric and Dimeric Forms of Recombinant Sml1p-histag Protein by Electrospray Mass Spectrometry

✍ Scribed by Tomoaki Uchiki; Robert Hettich; Vibha Gupta; Chris Dealwis

• Publisher: Elsevier Science
• Year: 2002
• Tongue: English
• Weight: 199 KB
• Volume: 301
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2001.5492

✦ Synopsis

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.