Department of Biochemistry, University of Sydney, NSW, 2006, Australia
Pulsed field gradient spin-echo (PFGSE) NMR spectroscopy is now generally regarded as the method of choice for measuring the translational diffusion coefficients of molecules of virtually any type under many conditions. For example it has been used to measure the translational diffusion of haemoglobin in intact human erythrocytes under high-resolution NMR conditions [1]. The spatial resolution of this technique is such that it can measure average molecular motion that occurs on a distance scale of nanometers; specifically, it can measure translation over distances of only twice the diameter of a haemoglobin molecule. Changes in the diffusion coefficient of a molecule provide a means for investigating small-molecule/macromolecule interactions; thus, we have measured the binding of 2,3-bisphosphoglycerate to haemoglobin in intact human erythrocytes in suspensions under states of different gas-binding [2].
The association of detergent molecules with a protein, used for its solubilisation for high resolution NMR analysis, can likewise be studied. Thus, the stoichiometry of the 'micelle' that forms between myosin light chain 2 and the cholate derivative CHAPS has been measured by using PFG NMR spectroscopy [3].
Since translational molecular motion is likely to be different inside and outside cells, in tissues, or in a cell suspension, transmembrane exchange of a solute will be manifest in an alteration in the measured apparent diffusion coefficient. Thus, measurements of diffusion coefficients of solutes and ions have been carried out in order to characterise membrane transport processes [4]. This transport is often protein mediated so the technique can yield information on the function as well as the structure of a transport/binding protein.
1. Kuchel, P. W., and Chapman, B. E. (1991) Translational Diffusion of Haemoglobin in Human Erythrocytes and Haemolysates. J. Magn. Reson. 94, 574-580.
2. Lennon, A.J., Scott, N.R., Chapman, B.E. and Kuchel, P.W. (1994) Haemoglobin Affinity for 2,3-bisphosphoglycerate in Solutions and Intact Erythrocytes: Studies Using Pulsed Field Gradient NMR and Monte Carlo Simulations. Biophys. J. 67, 2096-2109.
3. Dingley, A. J., Mackay, J. P., Chapman, B. E., Morris, M. B., Kuchel, P. W., Hambly, B. D., and King, G. F. (1995) Measuring Protein Self-Association using Pulsed-Field- Gradient NMR Spectroscopy: Application to Myosin Light Chain 2. (Submitted.)
4. Kuchel, P.W., Kirk, K, and King, G.F. (1994) NMR Methods for Measuring Membrane Transport. In: Physico-Chemical Methods in the Study of Biomembranes (Hilderson, H.J. and Ralston, G.B. eds). Chapter 7, Subcellular Biochemistry, 23, 247-327.