Towards miniaturization of a structural genomics pipeline using micro-expression and microcoil NMR

In structural genomics centers, nuclear magnetic resonance (NMR) screening is in increasing use as a tool to identify folded proteins that are promising targets for three-dimensional structure determination by X-ray crystallography or NMR spectroscopy. The use of 1D ¹H NMR spectra or 2D [¹H,¹⁵N]-correlation spectroscopy (COSY) typically requires milligram quantities of unlabeled or isotope-labeled protein, respectively. Here, we outline ways towards miniaturization of a structural genomics pipeline with NMR screening for folded globular proteins, using a high-density micro-fermentation device and a microcoil NMR probe. The proteins are micro-expressed in unlabeled or isotope-labeled media, purified, and then subjected to 1D ¹H NMR and/or 2D [¹H,¹⁵N]-COSY screening. To demonstrate that the miniaturization is functioning effectively, we processed nine mouse homologue protein targets and compared the results with those from the “macro-scale” Joint Center of Structural Genomics (JCSG) high-throughput pipeline. The results from the two pipelines were comparable, illustrating that the data were not compromised in the miniaturized approach.     

Optimization of resolution and sensitivity of 4D NOESY using Multi-dimensional Decomposition

Highly resolved multi-dimensional NOE data are essential for rapid and accurate determination of spatial protein structures such as in structural genomics projects. Four-dimensional spectra contain almost no spectral overlap inherently present in lower dimensionality spectra and are highly amenable to application of automated routines for spectral resonance location and assignment. However, a high resolution 4D data set using conventional uniform sampling usually requires unacceptably long measurement time. Recently we have reported that the use of non-uniform sampling and multi-dimensional decomposition (MDD) can remedy this problem. Here we validate accuracy and robustness of the method, and demonstrate its usefulness for fully protonated protein samples. The method was applied to 11 kDa protein PA1123 from structural genomics pipeline. A systematic evaluation of spectral reconstructions obtained using 15–100% subsets of the complete reference 4D ¹H–¹³C–¹³C–¹H NOESY spectrum has been performed. With the experimental time saving of up to six times, the resolution and the sensitivity per unit time is shown to be similar to that of the fully recorded spectrum. For the 30% data subset we demonstrate that the intensities in the reconstructed and reference 4D spectra correspond with a correlation coefficient of 0.997 in the full range of spectral amplitudes. Intensities of the strong, middle and weak cross-peaks correlate with coefficients 0.9997, 0.9965, and 0.83. The method does not produce false peaks. 2% of weak peaks lost in the 30% reconstruction is in line with theoretically expected noise increase for the shorter measurement time. Together with good accuracy in the relative line-widths these translate to reliable distance constrains derived from sparsely sampled, high resolution 4D NOESY data.     

蛋白质磁共振——从结构生物学到结构基因组学

在后基因组时代,随着大量物种全基因组序列的获得,结构生物学家面临着结构基因组学的新机遇和挑战。与传统的结构生物学不同的是,结构基因组学的研究主要集中在结构和功能未知并且与从前研究的蛋白质相似性很小的蛋白质。准确的来讲,结构基因组学通过高通量蛋白质表达、结构解析来完成所有蛋白质家族的结构表征,从而能够通过结构预测功能。加州结构基因组学联合实验室发展了高度自动化的蛋白质合成、结晶、结构解析生产线。然而由于一些蛋白质不能被结晶,要想覆盖所有蛋白质结构域还有很大困难。Wuthrich的研究小组通过一些高通量的目的蛋白质筛选和NMR结构解析的方法解决了这一难题。与X射线晶体学解析蛋白质结构相比,NMR技术由于能够解析更接近生理状态的溶液结构而具有互补性。通过获得溶液中的蛋白质稳定性、动力学特征和相互作用信息,正如在朊蛋白和SARS相关蛋白的研究中所表现的那样,NMR技术从扩大已知的蛋白质结构数据库、新的蛋白质功能到化学生物学研究中都扮演着激动人心的角色。     

A straight-forward method of optimising protein solubility for NMR

Maximising solubility is a key step in applying solution-state NMR to proteins. The microbatch crystallisation screening method can be adapted to screen for protein solubility. In this approach, drops of test solutions are placed under paraffin oil in 96-well screening plates. This requires very small amounts of protein, is easy to set up and is readily automatable.     

NMR Structure Determination and Structure-Based Functional Characterization of Conserved Hypothetical Protein MTH1175 from Methanobacterium thermoautotrophicum

The solution structure of MTH1175, a 124-residue protein from the archaeon Methanobacterium thermoautotrophicum has been determined by NMR spectroscopy. MTH1175 is part of a family of conserved hypothetical proteins (COG1433) with unknown functions which contains multiple paralogs from all complete archaeal genomes and the archaeal gene-rich bacterium Thermotoga maritima. Sequence similarity indicates this protein family may be related to the nitrogen fixation proteins NifB and NifX. MTH1175 adopts an α/β topology with a single mixed β-sheet, and contains two flexible loops and an unstructured C-terminal tail. The fold resembles that of Ribonuclease H and similar proteins, but differs from these in several respects, and is not likely to have a nuclease activity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Unique opportunities for NMR methods in structural genomics

This Perspective, arising from a workshop held in July 2008 in Buffalo NY, provides an overview of the role NMR has played in the United States Protein Structure Initiative (PSI), and a vision of how NMR will contribute to the forthcoming PSI-Biology program. NMR has contributed in key ways to structure production by the PSI, and new methods have been developed which are impacting the broader protein NMR community.