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. 2001 Dec;81(6):3156–3165. doi: 10.1016/S0006-3495(01)75952-8

The sensitivity of saturation transfer electron paramagnetic resonance spectra to restricted amplitude uniaxial rotational diffusion.

E J Hustedt 1, A H Beth 1
PMCID: PMC1301776  PMID: 11720982

Abstract

Computational methods have been developed to model the effects of constrained or restricted amplitude uniaxial rotational diffusion (URD) on saturation transfer electron paramagnetic resonance (ST-EPR) signals observed from nitroxide spin labels. These methods, which have been developed to model the global rotational motion of intrinsic membrane proteins that can interact with the cytoskeleton or other peripheral proteins, are an extension of previous work that described computationally efficient algorithms for calculating ST-EPR spectra for unconstrained URD (Hustedt and Beth, 1995, Biophys. J. 69:1409-1423). Calculations are presented that demonstrate the dependence of the ST-EPR signal (V'(2)) on the width (Delta) of a square-well potential as a function of the microwave frequency, the correlation time for URD, and the orientation of the spin-label with respect to the URD axis. At a correlation time of 10 micros, the V'(2) signal is very sensitive to Delta in the range from 0 to 60 degrees, marginally sensitive from 60 degrees to 90 degrees, and insensitive beyond 90 degrees. Sensitivity to Delta depends on the correlation time for URD with higher sensitivity to large values of Delta at the shorter correlation times, on the microwave frequency, and on the orientation of the spin-label relative to the URD axis. The computational algorithm has been incorporated into a global nonlinear least-squares analysis approach, based upon the Marquardt-Levenberg method (Blackman et al., 2001, Biophys. J. 81:3363-3376). This has permitted determination of the correlation time for URD and the width of the square-well potential by automated fitting of experimental ST-EPR data sets obtained from a spin-labeled membrane protein and provided a new automated method for analysis of data obtained from any system that exhibits restricted amplitude URD.

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Selected References

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  1. Barnes J. P., Liang Z., Mchaourab H. S., Freed J. H., Hubbell W. L. A multifrequency electron spin resonance study of T4 lysozyme dynamics. Biophys J. 1999 Jun;76(6):3298–3306. doi: 10.1016/S0006-3495(99)77482-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beth A. H., Venkataramu S. D., Balasubramanian K., Dalton L. R., Robinson B. H., Pearson D. E., Park C. R., Park J. H. 15N- and 2H-substituted maleimide spin labels: improved sensitivity and resolution for biological EPR studies. Proc Natl Acad Sci U S A. 1981 Feb;78(2):967–971. doi: 10.1073/pnas.78.2.967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blackman S. M., Cobb C. E., Beth A. H., Piston D. W. The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy. Biophys J. 1996 Jul;71(1):194–208. doi: 10.1016/S0006-3495(96)79216-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blackman S. M., Hustedt E. J., Cobb C. E., Beth A. H. Flexibility of the cytoplasmic domain of the anion exchange protein, band 3, in human erythrocytes. Biophys J. 2001 Dec;81(6):3363–3376. doi: 10.1016/S0006-3495(01)75969-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Casey J. R., Reithmeier R. A. Analysis of the oligomeric state of Band 3, the anion transport protein of the human erythrocyte membrane, by size exclusion high performance liquid chromatography. Oligomeric stability and origin of heterogeneity. J Biol Chem. 1991 Aug 25;266(24):15726–15737. [PubMed] [Google Scholar]
  6. Cuppoletti J., Goldinger J., Kang B., Jo I., Berenski C., Jung C. Y. Anion carrier in the human erythrocyte exists as a dimer. J Biol Chem. 1985 Dec 15;260(29):15714–15717. [PubMed] [Google Scholar]
  7. Howard E. C., Lindahl K. M., Polnaszek C. F., Thomas D. D. Simulation of saturation transfer electron paramagnetic resonance spectra for rotational motion with restricted angular amplitude. Biophys J. 1993 Mar;64(3):581–593. doi: 10.1016/S0006-3495(93)81417-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hustedt E. J., Beth A. H. Analysis of saturation transfer electron paramagnetic resonance spectra of a spin-labeled integral membrane protein, band 3, in terms of the uniaxial rotational diffusion model. Biophys J. 1995 Oct;69(4):1409–1423. doi: 10.1016/S0006-3495(95)80010-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hustedt E. J., Beth A. H. Determination of the orientation of a band 3 affinity spin-label relative to the membrane normal axis of the human erythrocyte. Biochemistry. 1996 May 28;35(21):6944–6954. doi: 10.1021/bi9601518. [DOI] [PubMed] [Google Scholar]
  10. Hustedt E. J., Cobb C. E., Beth A. H., Beechem J. M. Measurement of rotational dynamics by the simultaneous nonlinear analysis of optical and EPR data. Biophys J. 1993 Mar;64(3):614–621. doi: 10.1016/S0006-3495(93)81420-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hustedt E. J., Kirchner J. J., Spaltenstein A., Hopkins P. B., Robinson B. H. Monitoring DNA dynamics using spin-labels with different independent mobilities. Biochemistry. 1995 Apr 4;34(13):4369–4375. doi: 10.1021/bi00013a028. [DOI] [PubMed] [Google Scholar]
  12. Johnson M. E., Hyde J. S. 35-GHz (Q-band) saturation transfer electron paramagnetic resonance studies of rotational diffusion. Biochemistry. 1981 May 12;20(10):2875–2880. doi: 10.1021/bi00513a025. [DOI] [PubMed] [Google Scholar]
  13. Johnson M. E., Lee L., Fung L. W. Models for slow anisotropic rotational diffusion in saturation transfer electron paramagnetic resonance at 9 and 35 GHz. Biochemistry. 1982 Aug 31;21(18):4459–4467. doi: 10.1021/bi00261a041. [DOI] [PubMed] [Google Scholar]
  14. Jähnig F. The shape of a membrane protein derived from rotational diffusion. Eur Biophys J. 1986;14(1):63–64. doi: 10.1007/BF00260404. [DOI] [PubMed] [Google Scholar]
  15. Low P. S. Structure and function of the cytoplasmic domain of band 3: center of erythrocyte membrane-peripheral protein interactions. Biochim Biophys Acta. 1986 Sep 22;864(2):145–167. doi: 10.1016/0304-4157(86)90009-2. [DOI] [PubMed] [Google Scholar]
  16. Luna E. J., Hitt A. L. Cytoskeleton--plasma membrane interactions. Science. 1992 Nov 6;258(5084):955–964. doi: 10.1126/science.1439807. [DOI] [PubMed] [Google Scholar]
  17. Nigg E. A., Cherry R. J. Anchorage of a band 3 population at the erythrocyte cytoplasmic membrane surface: protein rotational diffusion measurements. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4702–4706. doi: 10.1073/pnas.77.8.4702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ryan T. A., Myers J., Holowka D., Baird B., Webb W. W. Molecular crowding on the cell surface. Science. 1988 Jan 1;239(4835):61–64. doi: 10.1126/science.2962287. [DOI] [PubMed] [Google Scholar]
  19. Saffman P. G., Delbrück M. Brownian motion in biological membranes. Proc Natl Acad Sci U S A. 1975 Aug;72(8):3111–3113. doi: 10.1073/pnas.72.8.3111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Squier T. C., Thomas D. D. Methodology for increased precision in saturation transfer electron paramagnetic resonance studies of rotational dynamics. Biophys J. 1986 Apr;49(4):921–935. doi: 10.1016/S0006-3495(86)83720-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Staros J. V., Kakkad B. P. Cross-linking and chymotryptic digestion of the extracytoplasmic domain of the anion exchange channel in intact human erythrocytes. J Membr Biol. 1983;74(3):247–254. doi: 10.1007/BF02332127. [DOI] [PubMed] [Google Scholar]
  22. Weinstein R. S., Khodadad J. K., Steck T. L. Fine structure of the band 3 protein in human red cell membranes: freeze-fracture studies. J Supramol Struct. 1978;8(3):325–335. doi: 10.1002/jss.400080310. [DOI] [PubMed] [Google Scholar]
  23. Wojcicki W. E., Beth A. H. Structural and binding properties of the stilbenedisulfonate sites on erythrocyte band 3: an electron paramagnetic resonance study using spin-labeled stilbenedisulfonates. Biochemistry. 1993 Sep 14;32(36):9454–9464. doi: 10.1021/bi00087a025. [DOI] [PubMed] [Google Scholar]

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