Fully automated assignment of methyl resonances of a 36 kDa protein dimer from sparse NOESY data

I Pritišanac,P Güntert,Jm Würz

doi:10.1088/1742-6596/1036/1/012008

Abstract

High-resolution solution-state NMR spectroscopy studies of large proteins typically require uniform deuteration of the system and selective protonation and isotope labelling of methyl groups. Under such circumstances, the assignment of methyl resonances presents a considerable experimental challenge and automation of the process using computational algorithms has been actively sought. Through-space connectivities between the labelled methyl groups can be established through nuclear Overhauser enhancement spectroscopy (NOESY). If a high-resolution structure of the system is available, the sparse connectivity restraints derived from this information enable structure-based methyl resonance assignment. Here, we outline a protocol for full automation of the methyl resonance assignment process using the CYANA software package. We tested the protocol on three-dimensional (3D) 13C/13C-separated NOESY spectra of a dimer of regulatory chains of aspartate transcarbamoylase (ATCase-r2). We used CYPICK to detect NOE signals, followed by automatic resonance assignment with FLYA. On this dataset, FLYA generated highly similar results using either automatically or manually generated peak lists, confidently assigning ∼60% of the methyl groups with high accuracy (95 ± 2% correctness). We compared this performance to two alternative automatic methyl assignment protocols, MAP-XSII and FLAMEnGO2.0, both of which, similarly to FLYA, support unassigned NOESY peak lists as input.

Highlights

High-resolution nuclear magnetic resonance (NMR) spectroscopy studies of biological macromolecules occupy a central role in biophysical chemistry, structural biology, drug design, structural genomics, and interactomics [1]
The CYPICK-generated nuclear Overhauser enhancement spectroscopy (NOESY) peak lists were directly fed to the structure-based FLYA assignment protocol. On this dataset, FLYA achieves similar levels of assignment coverage (~60%) and accuracy (~95%) as with manually prepared NOESY peak lists, and that these results compare favorably to alternative methyl resonance assignment approaches
It was previously shown that CYPICK performs well in combination with FLYA when applied to the assignment of backbone resonances of uniformly 15N/13C-labelled proteins with up to ~20 kDa molecular mass

Summary

Introduction

High-resolution nuclear magnetic resonance (NMR) spectroscopy studies of biological macromolecules occupy a central role in biophysical chemistry, structural biology, drug design, structural genomics, and interactomics [1]. Hydrogen (1H, or proton) is highly abundant in biological macromolecules and, owing to its high gyromagnetic ratio, is the most sensitive NMR probe of molecular structure and dynamics. Studies of complex biomolecules are aided by isotopic labelling that introduces NMR-active atomic nuclei to a system [2]. In studies of small and medium-size protein molecules, uniform labeling with carbon-13 (13C) and nitrogen-15 (15N) isotopes replaces NMR-inactive 12C and 14N nuclei. Cost-effective and robust biosynthetic strategies have been established for selective or simultaneous labelling of all methyl-containing amino acids in Escherichia coli [7,8], and of isoleucine-δ1 methyls in a eukaryote, Pichia pastoris [9]. One additional advantage of methyl groups is their high abundance in protein sequences, as six out of 20 amino acids have at least one methyl group in their side chain. Methyls are found both in the core and, to a lesser extent, at the surface of proteins, making them useful probes of protein structure and dynamics [10,11]

Methods

Results

Conclusion