Abstract
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 Sml1p-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.
| Original language | English |
|---|---|
| Pages (from-to) | 35-48 |
| Number of pages | 14 |
| Journal | Analytical Biochemistry |
| Volume | 301 |
| Issue number | 1 |
| DOIs | |
| State | Published - Feb 1 2002 |
Funding
We are grateful to Dr. Rodney Rothstein for kindly providing a plasmid containing Sml1p-histag ORF, PWJ750-2. T.U. acknowledges financial support from the Genome Science and Technology Graduate School, University of Tennessee–Oak Ridge National Laboratory. Dr. Gregory Hurst (ORNL) provided technical assistance for the MALDI-TOF experiments. 1Research was conducted under the Center for Structural and Molecular Biology Research Program at Oak Ridge National Laboratory and was sponsored by U.S. Department of Energy, Office of Biological and Environmental Research. Oak Ridge National Laboratory is operated and managed by University of Tennessee–Bat-telle, LLC, for the U.S. Department of Energy under Contract DE-AC05-00OR22725.
| Funders | Funder number |
|---|---|
| Genome Science and Technology Graduate School, University of Tennessee | |
| Office of Biological and Environmental Research | |
| U.S. Department of Energy | |
| Oak Ridge National Laboratory | |
| University of Tennessee | DE-AC05-00OR22725 |
Keywords
- Collisional dissociation
- Disulfide bond determination
- Electrospray
- Enzyme inhibition
- Mass spectrometry
- Proteolytic digests
- Recombinant protein