Quantitative Determination of Interacting Protein Surfaces in Prokaryotes and Eukaryotes by Using In-Cell NMR Spectroscopy.

Abstract

This paper describes three protocols for identifying interacting surfaces on 15N-labeled target proteins of known structure by using in-cell NMR spectroscopy. The first protocol describes how to identify protein quinary structure interaction surfaces in prokaryotes by using cross-relaxation-induced polarization transfer, CRIPT, based in-cell NMR. The second protocol describes how to introduce labeled protein into eukaryotic (HeLa) cells via electroporation for CRIPT-based in-cell studies. The third protocol describes how to quantitatively map protein interacting surfaces by utilizing singular value decomposition, SVD, analysis of STructural INTeractions by in-cell NMR, STINT-NMR, data.