Abstract
Direct fitting of sedimentation velocity data with numerical solutions of the Lamm equations has been exploited to obtain sedimentation coefficients for single solutes under conditions where solvent and solution plateaus are either not available or are transient. The calculated evolution was initialized with the first experimental scan and nonlinear regression was employed to obtain best-fit values for the sedimentation and diffusion coefficients. General properties of the Lamm equations as data analysis tools were examined. This method was applied to study a set of small peptides containing amphipathic heptad repeats with the general structure Ac-YS-(AKEAAKE)nGAR-NH2, n = 2, 3, or 4. Sedimentation velocity analysis indicated single sedimenting species with sedimentation coefficients (s(20,w) values) of 0.37, 0.45, and 0.52 S, respectively, in good agreement with sedimentation coefficients predicted by hydrodynamic theory. The described approach can be applied to synthetic boundary and conventional loading experiments, and can be extended to analyze sedimentation data for both large and small macromolecules in order to define shape, heterogeneity, and state of association.
Full Text
The Full Text of this article is available as a PDF (133.6 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Behlke J., Ristau O. Molecular mass determination by sedimentation velocity experiments and direct fitting of the concentration profiles. Biophys J. 1997 Jan;72(1):428–434. doi: 10.1016/S0006-3495(97)78683-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bloomfield V., Dalton W. O., Van Holde K. E. Frictional coefficients of multisubunit structures. I. Theory. Biopolymers. 1967 Feb;5(2):135–148. doi: 10.1002/bip.1967.360050202. [DOI] [PubMed] [Google Scholar]
- Byron O. Construction of hydrodynamic bead models from high-resolution X-ray crystallographic or nuclear magnetic resonance data. Biophys J. 1997 Jan;72(1):408–415. doi: 10.1016/S0006-3495(97)78681-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen Y. H., Yang J. T., Chau K. H. Determination of the helix and beta form of proteins in aqueous solution by circular dichroism. Biochemistry. 1974 Jul 30;13(16):3350–3359. doi: 10.1021/bi00713a027. [DOI] [PubMed] [Google Scholar]
- Claverie J. M., Dreux H., Cohen R. Sedimentation of generalized systems of interacting particles. I. Solution of systems of complete Lamm equations. Biopolymers. 1975 Aug;14(8):1685–1700. doi: 10.1002/bip.1975.360140811. [DOI] [PubMed] [Google Scholar]
- Cohen R., Claverie J. M. Sedimentation of generalized systems of interacting particles. II. Active enzyme centrifugation--theory and extensions of its validity range. Biopolymers. 1975 Aug;14(8):1701–1716. doi: 10.1002/bip.1975.360140812. [DOI] [PubMed] [Google Scholar]
- Cox D. J. Computer simulation of sedimentation in the ultracentrifuge. VI. Monomer-tetramer systems in rapid chemical equilibrium. Arch Biochem Biophys. 1971 Sep;146(1):181–195. doi: 10.1016/s0003-9861(71)80055-3. [DOI] [PubMed] [Google Scholar]
- Garcia de la Torre J., Navarro S., Lopez Martinez M. C., Diaz F. G., Lopez Cascales J. J. HYDRO: a computer program for the prediction of hydrodynamic properties of macromolecules. Biophys J. 1994 Aug;67(2):530–531. doi: 10.1016/S0006-3495(94)80512-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- García de la Torre J., Carrasco B., Harding S. E. SOLPRO: theory and computer program for the prediction of SOLution PROperties of rigid macromolecules and bioparticles. Eur Biophys J. 1997;25(5-6):361–372. doi: 10.1007/s002490050049. [DOI] [PubMed] [Google Scholar]
- Holladay L. A. An approximate solution to the Lamm equation. Biophys Chem. 1979 Sep;10(2):187–190. doi: 10.1016/0301-4622(79)85039-5. [DOI] [PubMed] [Google Scholar]
- Howlett G. J., Schachman H. K. Allosteric regulation of aspartate transcarbamoylase. Changes in the sedimentation coefficient promoted by the bisubstrate analogue N-(phosphonacetyl)-L-aspartate. Biochemistry. 1977 Nov 15;16(23):5077–5083. doi: 10.1021/bi00642a021. [DOI] [PubMed] [Google Scholar]
- Jackson R. L., Baker H. N., Gilliam E. B., Gotto A. M., Jr Primary structure of very low density apolipoprotein C-II of human plasma. Proc Natl Acad Sci U S A. 1977 May;74(5):1942–1945. doi: 10.1073/pnas.74.5.1942. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mulhern T. D., Howlett G. J., Reid G. E., Simpson R. J., McColl D. J., Anders R. F., Norton R. S. Solution structure of a polypeptide containing four heptad repeat units from a merozoite surface antigen of Plasmodium falciparum. Biochemistry. 1995 Mar 21;34(11):3479–3491. doi: 10.1021/bi00011a001. [DOI] [PubMed] [Google Scholar]
- Philo J. S. An improved function for fitting sedimentation velocity data for low-molecular-weight solutes. Biophys J. 1997 Jan;72(1):435–444. doi: 10.1016/S0006-3495(97)78684-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sartory W. K., Halsall H. B., Breillatt J. P. Simultation of gradient and band propagation in the centrifuge. Biophys Chem. 1976 Jul;5(1-2):107–135. doi: 10.1016/0301-4622(76)80029-4. [DOI] [PubMed] [Google Scholar]
- Stafford W. F., 3rd Boundary analysis in sedimentation transport experiments: a procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile. Anal Biochem. 1992 Jun;203(2):295–301. doi: 10.1016/0003-2697(92)90316-y. [DOI] [PubMed] [Google Scholar]
- Stafford W. F., 3rd Boundary analysis in sedimentation velocity experiments. Methods Enzymol. 1994;240:478–501. doi: 10.1016/s0076-6879(94)40061-x. [DOI] [PubMed] [Google Scholar]