An NMR approach to structural proteomics

Abstract

The influx of genomic sequence information has led to the concept of structural proteomics, the determination of protein structures on a genome-wide scale. Here we describe an approach to structural proteomics of small proteins using NMR spectroscopy. Over 500 small proteins from several organisms were cloned, expressed, purified, and evaluated by NMR. Although there was variability among proteomes, overall 20% of these proteins were found to be readily amenable to NMR structure determination. NMR sample preparation was centralized in one facility, and a distributive approach was used for NMR data collection and analysis. Twelve structures are reported here as part of this approach, which allowed us to infer putative functions for several conserved hypothetical proteins. S tructural proteomics, which aims to determine the threedimensional (3D) structures of all proteins, has become a major initiative within the biomedical community (see ref. 1 and other articles in the same issue). The large number of protein structures expected from these projects will yield valuable clues to the rules for predicting protein folding and understanding biochemical function. In these early stages of the structural proteomics effort, one of the main goals is to identify the best technologies and the most efficient processes to convert gene sequence into 3D structural information. One of the decisions will be to determine the optimal use of x-ray crystallography and NMR spectroscopy, which are the two techniques that will provide the majority of experimental data for these initiatives.X-ray crystallography currently is perceived as the potential workhorse for structural proteomics, because if provided with a well diffracting crystal it is possible to determine a 3D structure in hours. However, the throughput of structure determination using x-ray crystallography remains unclear, because the ratedetermining step continues to be the production of well diffracting crystals, a process that is unpredictable and can take between hours and months.NMR structure determination is limited currently by size constraints and lengthy data collection and analysis times (often months), and the method is best applied to proteins smaller than 250 amino acids. On the other hand, NMR experiments do not require crystals, and samples appropriate for structure determination can be identified within minutes of the protein being purified. In summary, x-ray crystallography and NMR spectroscopy seem to have complementary deficiencies, and the relative success of these methods in structural proteomics remains to be determined.We have shown previously that NMR spectroscopy can play a significant role in structural proteomics even with its current limitations (2). The initial pilot project, based on a limited number of proteins from the thermophilic archaebacterium Methanobacterium thermoautotrophicum (Mth) suggested that smaller proteins may be more amenable to structure analysis, because in this genome a higher proportion of smaller proteins were soluble compar...

AutoLink: Automated sequential resonance assignment of biopolymers from NMR data by relative-hypothesis-prioritization-based simulated logic

“…The data were obtained from BMRB Accession Nos. 4678 [26], 5106 [27], 5166 [27], 5329 [28], 5335 [29], 5589 [30], 5656 [31], 5691 [32], 5842 [33], 5844 [34], 5845 [35], 5859 [36], 6011 [37], 6052 [38], 6128 [39], 6138 [40], 6176 [41], 6209 [42], 6318 [43], 6341 [44], and 6344 [45]. Of these sets, the peak information was available from 6128, 6176, and 6318.…”

ABACUS, a direct method for protein NMR structure computation via assembly of fragments

“…Uniformly 13 C/ 15 N-labeled mth1743 was expressed and purified as previously described. 6 The purified protein contained the complete sequence of mth1743 plus three additional N-terminal residues (Gly-Ser-His) remaining after proteolytic cleavage of the His6 affinity tag. The concentration of protein samples ranged from 1.0 to 1.5 mM in an aqueous solution containing 25 mM sodium phosphate (pH 6.5), 550 mM NaCl, 1 mM DTT, 95% H 2 O/5% D 2 O.…”

“Fuzzy oil drop” model applied to individual small proteins built of 70 amino acids

“…The proteins chosen for analysis and presented here are a subset of the selected entries and belong to one of the following functional groups: growth factors, serine protease inhibitors, antifreeze proteins, chaperones and proteins of unknown function (Table 1). The data for the analyzed structures were taken from investigations carried out using X-ray crystallography [26][27][28] and NMR techniques [29][30][31][32][33][34][35][36][37][38][39][40]. Additionally, the SH3 domain is discussed with respect to the various proteins that contain it and their different biological functions [41].…”