Unknown

Dataset Information

0

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


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 cross-linker 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.

SUBMITTER: Zybailov B 

PROVIDER: S-EPMC4722957 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

altmetric image

Publications

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

Zybailov Boris B   Gokulan Kuppan K   Wiese Jadon J   Ramanagoudr-Bhojappa Ramanagouda R   Byrd Alicia K AK   Glazko Galina G   Jaiswal Mihir M   Mackintosh Samuel S   Varughese Kottayil I KI   Raney Kevin D KD  

Journal of proteomics & bioinformatics 20151119 11


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 cross-linker or formaldehyde captured the interaction between recombinant Rim1 and Pif1. In addition, Pif1 needed to be stripped of its N-terminal and C-termi  ...[more]

Similar Datasets

| S-EPMC3553982 | biostudies-literature
| S-EPMC5981002 | biostudies-literature
| S-EPMC4756656 | biostudies-literature
| S-EPMC5321032 | biostudies-literature
| S-EPMC3086679 | biostudies-literature
| S-EPMC7338081 | biostudies-literature
| S-EPMC2938055 | biostudies-literature
| S-EPMC4084482 | biostudies-literature
| S-EPMC4100091 | biostudies-literature
| S-EPMC3931034 | biostudies-literature