Determination of Oligomeric State of Proteins in Solution from Pulsed-Field-Gradient Self-Diffusion Coefficient Measurements. A Comparison of Experimental, Theoretical, and Hard-Sphere Approximated Values
✍ Scribed by V.V. Krishnan
- Publisher
- Elsevier Science
- Year
- 1997
- Tongue
- English
- Weight
- 131 KB
- Volume
- 124
- Category
- Article
- ISSN
- 1090-7807
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✦ Synopsis
The first step toward protein structure determination using of self-diffusion coefficient in peptides (17,18), proteins NMR is elucidation of the appropriate solution conditions (9-14), and DNA oligomers (15). Altieri et al. (12) have that obviate any protein self-association at the millimolar recently adopted the LED (longitudinal encode-decode) concentration necessary for the analysis (1). Knowledge on experiment proposed by Gibbs and Johnson (19), with an the oligomerization of the protein prior to the data collection improved water suppression scheme, to measure self-diffuis rather important, due to the complexity associated with sion coefficients of proteins. Ratio of the experimentally assigning and identifying long-range nuclear Overhauser efmeasured diffusion constants was used in comparison with fect (NOE) contacts in the process of determining the threethe expected ratio for a hard-sphere contact of monomers to dimensional structure (2). In general, the oligomerization determine the oligomerization state of proteins (9, 12). This state of a protein of interest is characterized using biophysimethod provides a quick way to measure the oligomerization cal methods like equilibrium ultracentrifugation or dynamic state of proteins under the NMR sample conditions. light scattering measurements (3). Even though reliable re-
The purpose of the present note is to evaluate how well sults are obtained in most cases, these measurements are the hard-sphere approximation of dimer formation is adapted done at a much lower protein concentration, which differs in determining the oligomerization state of proteins. This is about three orders of magnitude compared to the sample achieved by measuring the self-diffusion coefficient expericoncentration for the NMR experiments. Hence, determinamentally, using the LED sequence, as well as calculating it tion of the oligomerization state of the protein under the theoretically using the beads theory of GarcıB a de la Torre sample conditions of the NMR experiments is primarily im-(20) on a set of proteins of known oligomerization state. portant.
When these results are compared with the ratios obtained The use of pulsed magnetic field gradients in high-resoluby approximating the formation of dimers as hard-sphere tion NMR is a major advancement due to the availability monomer contacts, only a qualitative agreement is obtained. of self-shielded gradient coils in commercial spectrometers Experimentally measured self-diffusion coefficients show a (4, 5). Among the many potential applications of pulsed larger dispersion with the ideal molecular weights obtained field gradients (PFG) are the reduction in the number of from amino-acid composition, compared to the calculated phase-cycling steps needed for a specific coherence-transfer values. The origin of such a difference is ascribed to the pathway and efficient cancellation of spectral artifacts interaction of the protein with the solvent medium. It has (6, 7). In addition to the modern applications of PFG in also been found that self-diffusion coefficients obtained exbiomolecular NMR (6, 7), classical experimental methods perimentally as well theoretically correlate linearly with the to measure self-diffusion coefficients using PFG (8) have solvent-accessible surface area (21,22), providing an addibeen applied to proteins and DNA oligomers to understand tional parameter to represent quaternary structure of proteins. the molecular association processes (9-18).
The basis of the LED-PFG sequence is the use of bipolar PFG NMR has been widely used to measure tracer diffugradient pulses (19,23,24) to minimize eddy-current effects. sion coefficients of small molecules and polymers in solution As in the conventional stimulated-echo experiment of Tanner (16). However, only a few reports describe the measurement (25), a pair of matched gradient pulses are separated by the interval (D 0 d), each having an effective area of q, where * Currrent address: Biology and Biotechnology Research Program, Lawq is a product of gg z d, with g the gyromagnetic ratio of protons,