Ramanagouda Ramanagoudr-Bhojappa
Tanzania
Research Article
Analysis of Protein-protein Interaction Interface between Yeast Mitochondrial Proteins Rim1 and Pif1 Using Chemical Cross-linking Mass Spectrometry
Author(s): Boris Zybailov, Kuppan Gokulan, Jadon Wiese, Ramanagouda Ramanagoudr-Bhojappa, Alicia K. Byrd, Galina Glazko, Mihir Jaiswal, Samuel Mackintosh, Kottayil I. Varughese and Kevin D. RaneyBoris Zybailov, Kuppan Gokulan, Jadon Wiese, Ramanagouda Ramanagoudr-Bhojappa, Alicia K. Byrd, Galina Glazko, Mihir Jaiswal, Samuel Mackintosh, Kottayil I. Varughese and Kevin D. Raney
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.. View More»
DOI: 10.4172/jpb.1000376
