Accelerating NMR-Based Structural Studies of Proteins by Combining Amino Acid Selective Unlabeling and Fast NMR Methods

Bankala Krishnarjuna,Kousik Chandra,Hanudatta Atreya

doi:10.3390/magnetochemistry4010002

Bankala Krishnarjuna, Kousik Chandra + Show 1 more

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https://doi.org/10.3390/magnetochemistry4010002

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Abstract

In recent years, there has been a growing interest in fast acquisition and analysis of nuclear magnetic resonance (NMR) spectroscopy data for high throughput protein structure determination. Towards this end, rapid data collection techniques and methods to simplify the NMR spectrum such as amino acid selective unlabeling have been proposed recently. Combining these two approaches can speed up further the structure determination process. Based on this idea, we present three new two-dimensional (2D) NMR experiments, which together provide 15N, 1HN, 13Cα, 13Cβ, 13C′ chemical shifts for amino acid residues which are immediate C-terminal neighbors (i + 1) of residues that are selectively unlabeled. These experiments have high sensitivity and can be acquired rapidly using the methodology of G-matrix Fourier transform (GFT) NMR spectroscopy combined with non-uniform sampling (NUS). This is a first study involving the application of fast NMR methods to proteins samples prepared using a specific labeling scheme. Taken together, this opens up new avenues to using the method of selective unlabeling for rapid resonance assignment of proteins.

Highlights

During the last decade, there has been a growing emphasis on speeding up the structure determination process of proteins by nuclear magnetic resonance (NMR) spectroscopy, especially in the context of structural genomics projects, which require high throughput structure determination [1–4]
The application of two fast NMR methods, namely G-matrix Fourier transform (GFT) NMR and non-uniform sampling to selectively unlabeled protein samples expands the repertoire of applications possible with selective unlabeling
Three new NMR experiments are presented which are applicable to protein samples prepared with amino acid selective unlabeling

Summary

Introduction

There has been a growing emphasis on speeding up the structure determination process of proteins by NMR spectroscopy, especially in the context of structural genomics projects, which require high throughput structure determination [1–4]. This is due to the fact that the conventional approaches for NMR data acquisition and analysis are time consuming. Selective unlabeling or ”reverse” labeling involves the 13C/15N enrichment of all but specific chosen amino acids in a protein, which are rendered unlabeled (12C/14N) [11,13–17] This helps in simplifying the NMR spectrum by reducing the number of peaks in the spectrum, and thereby, aiding unambiguous resonance assignments

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