Determination of solution structures of proteins up to 40 kDa using CS-Rosetta with sparse NMR data from deuterated samples

Abstract

We have developed an approach for determining NMR structures of proteins over 20 kDa that utilizes sparse distance restraints obtained using transverse relaxation optimized spectroscopy experiments on perdeuterated samples to guide RASREC Rosetta NMR structure calculations. The method was tested on 11 proteins ranging from 15 to 40 kDa, seven of which were previously unsolved. The RASREC Rosetta models were in good agreement with models obtained using traditional NMR methods with larger restraint sets. In five cases X-ray structures were determined or were available, allowing comparison of the accuracy of the Rosetta models and conventional NMR models. In all five cases, the Rosetta models were more similar to the X-ray structures over both the backbone and side-chain conformations than the "best effort" structures determined by conventional methods. The incorporation of sparse distance restraints into RASREC Rosetta allows routine determination of high-quality solution NMR structures for proteins up to 40 kDa, and should be broadly useful in structural biology.nuclear magnetic resonance | sparse data | maltose binding protein | structural genomics | genetic algorithms A dvances in hardware, sample preparation, pulse sequence development, and refinement techniques have expanded the size and complexity of proteins accessible to structure determination by solution-state NMR to include proteins that, until recently, were exclusively the realm of X-ray crystallography (1-3). However, despite a number of landmark studies (4-7), only a small percentage of structures solved by NMR and deposited in the Protein Data Bank exceed 20 kDa in molecular weight. Larger structures need to be assembled by combining structural information from individual domains, and require additional techniques to elucidate the spatial arrangement, such as shape fitting (5) and/or paramagnetic restraints (8).The 20-kDa general limit coincides with the two fundamental problems in solution-state NMR: resonance overlap and progressive increase in the transverse relaxation rate (1∕T 2 ). As the size of a molecule increases, so does the rotational correlation time and, consequently, the efficiency of 1 H-1 H relaxation mechanisms. One way to suppress these effects is to incorporate deuterium into the protein sample, diluting the 1 H-1 H relaxation networks and increasing 13 C and 15 N relaxation times, resulting in sharper line widths and dramatic improvement of the signalto-noise ratios (2, 9, 10). Perdeuteration is generally required for studies of larger proteins (11-14), particularly membrane proteins (15, 16).Unfortunately, deuteration also eliminates the majority of 1 H-1 H NOEs, the main source of long-range distance information in solution-state NMR. Several methods have emerged for reintroducing protons at selected sites to function as distance probes in the structure (11,17). Methyl groups of isoleucine δ1, leucine, and valine side chains are straightforward to label with 13 C and 1 H isotopes in an otherwise deuterated protein sample (12, ...

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