alexa Analysis of Protein-protein Interaction Interface between Yeast Mitochondrial Proteins Rim1 and Pif1 Using Chemical Cross-linking Mass Spectrometry | OMICS International
ISSN: 0974-276X

Journal of Proteomics & Bioinformatics
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Research Article

Analysis of Protein-protein Interaction Interface between Yeast Mitochondrial Proteins Rim1 and Pif1 Using Chemical Cross-linking Mass Spectrometry

Boris Zybailov1*, Kuppan Gokulan4, Jadon Wiese1, Ramanagouda Ramanagoudr-Bhojappa1, Alicia K. Byrd1, Galina Glazko2, Mihir Jaiswal3, Samuel Mackintosh1, Kottayil I. Varughese4 and Kevin D. Raney1*

1Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA

2Department of Biomedical Informatics, University of Arkansas for Medical Sciences, USA

3UALR/UAMS joint bioinformatics program, University of Arkansas Little Rock, Little Rock, AR, USA

4Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR-72205, USA

*Corresponding Authors:
Boris Zybailov
4301 W Markham, Mail Slot #516
Little Rock, AR, 72205, USA
Tel/Fax: (501)686-8169
E-mail: [email protected]
 
Kevin D. Raney
Department of Biochemistry and Molecular Biology
University of Arkansas for Medical Sciences
Little Rock, AR, USA
E-mail: [email protected]

Received date: August 05, 2015; Accepted date: November 16, 2015; Published date: November 19, 2015

Citation: Zybailov B, Gokulan K, Wiese J, Ramanagoudr-Bhojappa R, Byrd AK, et al. (2015) Analysis of Protein-protein Interaction Interface between Yeast Mitochondrial Proteins Rim1 and Pif1 Using Chemical Cross-linking Mass Spectrometry. J Proteomics Bioinform 8: 243-252. doi: 10.4172/jpb.1000376

Copyright: © 2015 Zybailov B, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Defining protein-protein contacts is a challenging problem and cross-linking is a promising solution. Here, we present a case of mitochondrial single strand binding protein Rim1 and helicase Pif1, an interaction first observed in immuno-affinity pull-down from yeast cells using Pif1 bait. We found that only the short succinimidyl-diazirine crosslinker or formaldehyde captured the interaction between recombinant Rim1 and Pif1. In addition, Pif1 needed to be stripped of its N-terminal and C-terminal domains, and Rim1’s C-terminus needed to be modified for the cross-linked product to become visible. Our report is an example of a non-trivial analysis, where a previously identified stable interaction escapes initial capture with cross-linking agents and requires substantial modification to recombinant proteins and fine-tuning of the mass spectrometry-based methods for the cross-links to become detectable.

We used high resolution mass spectrometry to detect the cross-linked peptides. A 1:1 mixture of 15N and 14N-labeled Rim1 was used to validate the cross-links by their mass shift in the LC-MS profiles. Two sites on Rim1 were confirmed: 1) the N-terminus, and 2) the K29 residue. Performing cross-linking with a K29A variant visibly reduced the cross-linked product. Further, K29A-Rim1 showed a five-fold lower affinity to single stranded DNA compared to wild-type Rim1. Both the K29A variant and wild type Rim1 showed similar degrees of stimulation of Pif1 helicase activity. We propose structural models of the Pif1-Rim1 interaction and discuss its functional significance. Our work represents a non-trivial protein-protein interface analysis and demonstrates utility of short and non-specific cross-linkers.

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