An NMR approach to structural genomics

Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, 101 College Street, Toronto, ON, Canada M5G 1L7.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2002; 99(4):1825-30. DOI: 10.1073/pnas.042684599
Source: PubMed

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.

Download full-text


Available from: Antonio Pineda-Lucena, Sep 28, 2015
120 Reads
  • Source
    • "While a couple of these ''missing'' amide cross peaks may exist underneath assigned residues (with carbon chemical shift overlap in the backbone 3D experiments as well), the clustering of missing amide resonances in two regions near the N-terminus suggests that most of these ''missing'' residues are truly absent. Various strategies were unsuccessfully employed to observe more of the sRisS resonances in the 1 H– 15 N HSQC spectrum (Yee et al. 2002). Increasing the temperature from 303 to 318 K produced sharper spectra but no additional amide cross peaks while lowering the temperature from 303 to 273 K resulted in a loss of resolution and the ''merging'' of resonances due to line broadening. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Using a deuterated sample, all the observable backbone (1)H(N), (15)N, (13)C(a), and (13)C' chemical shifts for the dimeric, periplasmic sensor domain of the Burkholderia pseudomallei histidine kinase RisS were assigned. Approximately one-fifth of the amide resonances are "missing" in the (1)H-(15)N HSQC spectrum and map primarily onto α-helices at the dimer interface observed in a crystal structure suggesting this region either undergoes intermediate timescale motion (μs-ms) and/or is heterogeneous.
    Biomolecular NMR Assignments 05/2015; 9(2). DOI:10.1007/s12104-015-9614-2 · 0.76 Impact Factor
  • Source
    • "Investigations on improving of kinetic activity and stability have been the topics of active research, but study for increasing protein solubility receives less attention. Cumulative documents reveal that up to 80% of the identified non-membrane proteins are low protein solubility [1] [2] [3]. This problem often obstructs the preparation of concentrated protein for high-resolution structural determination, quantitative binding assays, and characterization of the novel proteins. "
    [Show abstract] [Hide abstract]
    ABSTRACT: From research to application, high protein solubility is usually a desired property yet sometimes difficult to achieve. The in vitro low solubility of the fully folded proteins is relevant to applied microbiological studies, biochemical studies, biopharmaceutical studies, high-resolution structural studies, and applications demanding high protein concentration. This insufficient protein solubility depends largely on the surface property of the protein molecule. To alleviate this problem, approaches emphasized on the improvement of water-binding ability or prevention of protein aggregation were employed including the use of chemical additives, fusion with solubility enhancement tags, and molecular engineering of the surface amino acid residues. With the availability of the three-dimensional structure of the target proteins, the effect of different surface amino acid residues on protein solubility could be systematically investigated. With the applications of advanced bioinformatics tools and guided by protein three-dimensional structure, solubility-improving mutagenesis can be designed and executed with a high chance of success. Integrating rational molecular engineering with other available approaches will be the effective strategy for alleviating in vitro low solubility of important proteins in the future.
    02/2015; 8(2):137-143. DOI:10.14416/j.ijast.2014.12.003
  • Source
    • "When used in conventional context, NMR spectroscopy often represents a major time bottleneck for sampling adequate number of points for ultimate success of peaks resolution [7], [8]. The digital resolution in the frequency domain refers to the minimum separation between two adjacent points. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Adequate digital resolution and signal sensitivity are two critical factors for protein structure determinations by solution NMR spectroscopy. The prime objective for obtaining high digital resolution is to resolve peak overlap, especially in NOESY spectra with thousands of signals where the signal analysis needs to be performed on a large scale. Achieving maximum digital resolution is usually limited by the practically available measurement time. We developed a method utilizing non-uniform sampling for balancing digital resolution and signal sensitivity, and performed a large-scale analysis of the effect of the digital resolution on the accuracy of the resulting protein structures. Structure calculations were performed as a function of digital resolution for about 400 proteins with molecular sizes ranging between 5 and 33 kDa. The structural accuracy was assessed by atomic coordinate RMSD values from the reference structures of the proteins. In addition, we monitored also the number of assigned NOESY cross peaks, the average signal sensitivity, and the chemical shift spectral overlap. We show that high resolution is equally important for proteins of every molecular size. The chemical shift spectral overlap depends strongly on the corresponding spectral digital resolution. Thus, knowing the extent of overlap can be a predictor of the resulting structural accuracy. Our results show that for every molecular size a minimal digital resolution, corresponding to the natural linewidth, needs to be achieved for obtaining the highest accuracy possible for the given protein size using state-of-the-art automated NOESY assignment and structure calculation methods.
    PLoS ONE 07/2013; 8(7):e68567. DOI:10.1371/journal.pone.0068567 · 3.23 Impact Factor
Show more