Technical Highlight - September 2009
Short description: With the time needed to solve structures by NMR spectroscopy reduced to weeks or even days, how much better can NMR get?
Well-behaved small-to-medium sized proteins (8–25 kDa) can be solved within just a few days using NMR spectroscopy. The average time to produce a structure using high-throughput techniques is now two to three weeks, and the structures that have been produced include many classes of proteins, including enzymes. This impressive increase in speed goes hand in hand with improved sensitivity, allowing structures to be achieved from mere micrograms of purified protein. How have these improvements been achieved and what else can we look forward to?
Some of these advances have come through high-throughput methods developed by the Protein Structure Initiative. For example, the three PSI centers that routinely use NMR spectroscopy — NESG, CESG and JCSG — have not only improved the output time but also substantially lowered cost per structure. Other exciting developments have been made by individual laboratories pursing new approaches, such as in-cell NMR.
Time and cost savings have been achieved partly through use of the eukaryotic wheat-germ cell-free expression system and condensed-phase bacterial single protein production (cSPP) systems to isotope-enrich proteins for spectroscopy. In addition, the development of micro-coil NMR probes that can produce three-dimensional structures using samples containing less than 100 micrograms of protein has opened up NMR to proteins that can only be produced in limited amounts.
The use of cryogenic NMR probes, providing more than a factor of 10 improvement in signal-to-noise ratios, together with methods of NMR data collection designed to exploit this higher sensitivity, for example, G-matrix Fourier transform NMR, has reduced NMR data-collection times by more than an order of magnitude. Structure determinations are also carried out in a largely automated fashion, using software for data analysis, structure calculations and structure-quality checking.
But what further developments can we expect? An important advantage of NMR is its ability to provide a dynamic view of proteins, which will be particularly useful for examining natively unfolded proteins, functionally important flexible surface loops and enzyme active sites. At present it is the only technique that can detect weak transient structural interactions, and it is useful for studying functionally important protein–protein and protein–ligand interactions.
Another exciting area ripe for development is in-cell NMR, which exploits NMR's ability to study proteins in their natural cellular environment. In this new way, NMR will also be systematically used to combine experimental protein binding, protein dynamics, and/or biochemical information with structural data. All of these developments are likely to become more widely available.
But despite these advances in NMR spectroscopy, obtaining suitable pure protein remains the most expensive and time-consuming step in the production of a three-dimensional protein structure. Improving expression, solubility and homogeneity remains key to NMR success.
G. T. Montelione et al. Unique opportunities for NMR methods in structural genomics.
J. Struct. Funct. Genomics 10, 101-106 (2009). doi:10.1007/s10969-009-9064-0
J. M. Aramini et al. Solution NMR Structure of the NlpC/P60 Domain of Lipoprotein Spr from Escherichia coli: Structural Evidence for a Novel Cysteine Peptidase Catalytic Triad.
Biochemistry 47, 9715-9717 (2008). doi:10.1021/bi8010779