Dynamic Light Scattering

Dynamic light scattering (also known as photon correlation spectroscopy) from particles in solution can be used to deduce information about the size, shape, and dynamics of biological macromolecules and supramolecular assemblies. Structural analysis of individual biomolecules, conformational transitions, and intermolecular interactions can also be probed. The major use of dynamic light scattering in biology is the rapid determination of the translational diffusion constant for macromolecules in solution. The interpretation of diffusion constant data is frequently strengthened by combination with other information from methods, such as electron microscopy, static light scattering, and neutron or X-ray small-angle scattering. Biological systems are well-suited to study by dynamic light scattering, because they are generally large enough to be strong scatterers at low concentrations and their diffusion constants are such that they give rise to autocorrelation functions that can be readily measured. Dynamic light scattering measurements have been used for studying objects ranging in size from proteins as small as 6000 Da to intact cells as large as micrometers.

 Theory of Dynamic Light Scattering

Dynamic light scattering measures light scattering intensity fluctuations in a small volume of a sample. For small proteins ( ~25kDa) these fluctuations are on the microsecond timescale, while for cells they are in milliseconds. The fluctuations are related to the Brownian motion of the particles giving rise to density fluctuations caused by variations in the number of molecules in the scattering volume and random agglomerations. This measurement in the time domain is related to the spectral density of the fluctuations in the frequency domain by a Fourier transformation. In practice, diffusion coefficients are determined using an autocorrelation function that is measured by accumulating the product of the number of photons arriving at the detector from successive time intervals. This operation is repeated thousands of times and averaged. The intensity autocorrelation function, G₂( t), is obtained by storing the average products I_t′I_t+t′, where t is an incremented time delay, in successive channels to yield

For a solution of macromolecules, assuming a Gaussian distribution of fluctuations, G₂(t) is related to the scattered electric field autocorrelation function G₁(t) by 

For translation diffusion of monodisperse particles that are small with respect to the incident wavelength, l: 

where A and B are constants that depend upon experiment geometry, and 

D is the translational diffusion coefficient, and G is the reciprocal of the characteristic decay time,

where O is the scattering angle. For a continuous polydisperse system, equation (4) is integrated over all sizes, and hence G values, to give

where G(G) is a distribution function that can be evaluated.

 Rotational diffusion and internal dynamics can also influence the autocorrelation functions measured in a dynamic light scattering experiments. The internal dynamics of a macromolecule in solution become important only if the amplitude of the motions is not very much smaller than the wavelength of the light. For large motions, different parts of the sample molecule will scatter out of phase, and the internal dynamics will contribute one or more relaxation times that will contribute to the decay of the autocorrelation function in a single or multiple-exponential fashion.

Examples of Dynamic Light Scattering Applications 

The transcription, translation, and replication of genetic information contained in the sequences of DNA is controlled by interactions of proteins with DNA and with RNA. These interactions can be probed using dynamic light scattering. For example, the enzyme DNA gyrase catalyzes the ATP-dependent supercoiling of DNA. The diffusion constant for DNA gyrase measured by dynamic light scattering (1) is significantly smaller than expected based on its molecular weight, and binding of DNA to the gyrase produces no change in the diffusion constant, indicating that there is no overall conformational change. These observations, combined with parallel small-angle neutron scattering experiments to determine radius of gyration values for the gyrase and its complex with DNA, have led to the conclusion that the gyrase has grooves on its surface to accommodate the DNA in a very compact complex.

Dynamic light scattering has played a role in drug development. The pharmacological effects of drugs can depend upon their behavior in solution and the characteristics of aggregates they may form (2). Lipids can be used to “solubilize” drugs that may be insoluble in aqueous media, and hence enhance their transport and efficacy. Dynamic light scattering has been used to evaluate aggregation behavior of antidepressants (3), critical micelle concentrations, and sizes of micelles designed to encapsulate the drug Indomethecin (4), as well as to evaluate how the opiate drug loperamide alters the temperature-induced phase transition of phosphatidylcholine vesicles (5).

Dynamic light scattering has also been used to monitor the assembly of enveloped viruses. Lyles et al. (6) used a combination of dynamic and static light scattering with stopped flow to study matrix protein binding to nucleocapsids of vesicular stomatitis virus. Dynamic light scattering has recently become a common tool for assessing the crystallizability of macromolecules and macromolecular assemblies (7, 8). Because modern molecular biology techniques have provided sufficient amounts of pure materials, dynamic light scattering can be used automatically to “screen” large numbers of crystallization conditions to determine diffusion constants and hence evaluate possible aggregation states that inhibit crystallization.

References

1. S. Krueger et al. (1990) J. Mol Biol. 211, 211–220. 

2. D. Attwood and P. Fletcher (1986) J. Pharm. Pharmacol. 38, 494–498.

3. A. D. Atherton and B. W. Barry (1985) J. Pharm. Pharmacol. 37, 854–862. 

4. D. Attwood, G. Ktistis, Y. McCormick, and M. J. Story (1989) J. Pharm. Pharmacol. 41, 83–86. 

5. E. Hantz, A. Cao, R. S. Phadke, and E. Taillandier (1989) Chem. Phys. Lipids 51, 75–82. 

6. D. S. Lyles, M. O. McKenzie, and R. R. Hangton (1996) Biochemistry 35, 6508–6518. 

7. A. D''Arcy (1994) Acta Crystallogr. D50, 469–471. 

8. A. Ferre-D‘Amare and S. K. Burley (1994) Structure (London) 2, 357–359.