Share This Article:

Monitoring the autoproteolysis of hiv-1 protease by site-directed spin-labeling and electron paramagnetic resonance spectroscopy

Abstract Full-Text HTML Download Download as PDF (Size:822KB) PP. 137-146
DOI: 10.4236/jbpc.2011.22017    5,391 Downloads   10,151 Views   Citations

ABSTRACT

Site-directed spin-labeling with continuous wave electron paramagnetic resonance spectroscopy was used to monitor autoproteolysis of HIV-1 protease, an enzyme essential for viral maturation. Two protein constructs were examined, namely subtype F and the circulating recombinant form CRF01_A/E. As the protease undergoes self-cleavage, protein unfolds and small peptide fragments containing the spin label are generated, which collectively give rise to a sharp spectral component that is easily discernable in the high-field resonance line in the EPR spectrum. By monitoring the intensity of this spectral component over time, the autoproteolytic stability of each construct was characterized under various conditions. Data were collected for samples stored at 4 °C, 25 °C, and 37 °C, and on a subtype F HIV-1 protease sample stored at 25 °C and containing the FDA-approved protease inhibitor Tipranavir. As expected, the rate of autoproteolysis decreased as the storage temperature was lowered. Minimal autoproteolysis was seen for the sample that contained Tipranavir, providing direction for future spectroscopic studies of active protease samples. When compared to standard methods of monitoring protein degradation such as gel electrophoresis or chromatographic analyses, spin-labeling with CW EPR offers a facile, real-time, non-consuming way to monitor autoproteolysis or protein degradation. Additionally, mass spectrometry studies revealed that the N-termini of both constructs are sensitive to degradation and that the sites of specific autoproteolysis vary.

Conflicts of Interest

The authors declare no conflicts of interest.

Cite this paper

Kear, J. , Galiano, L. , Veloro, A. , Busenlehner, L. and Fanucci, G. (2011) Monitoring the autoproteolysis of hiv-1 protease by site-directed spin-labeling and electron paramagnetic resonance spectroscopy. Journal of Biophysical Chemistry, 2, 137-146. doi: 10.4236/jbpc.2011.22017.

References

[1] P. Ashorn, T. J. McQuade, S. Thaisrivongs, A. G. Tomasselli, W. G. Tarpley, B. Moss, "An inhibitor of the protease blocks maturation of human and simian immunodeficiency viruses and spread of infection," Proceedings of the National Academy of Sciences USA, Vol. 87, num. 19, October 1990, pp. 7472-7476. doi:10.1073/pnas.87.19.7472
[2] J. O. Hui, A. G. Tomasselli, I. M. Reardon, J. M. Lull, D. P. Brunner, C. S. Tomich, R. L. Heinrikson, "Large scale purification and refolding of HIV-1 protease from Escherichia coli inclusion bodies," Journal of Protein Chemistry, Vol. 12, num. 3, June 1993, pp. 323-327. doi:10.1007/BF01028194
[3] J. R. Rosé, R. Salto, S. Craik, "Regulation of autoproteolysis of the HIV-1 and HIV-2 proteases with engineered amino acid substitutions," Journal of Biological Chemistry, Vol. 268, num. 16, June 1993, pp. 11939-11945.
[4] J. E. Strickler, J. Gorniak, B. Dayton, T. Meek, M. Moore, V. Magaard, J. Malinowski, C. Debouck, "Characterization and autoprocessing of precursor and mature forms of human immunodeficiency virus type 1 (HIV-1) protease purified from Escherischia coli," Proteins: Structure, Function, and Bioinformatics, Vol. 6, num. 2, 1989, pp. 139-154.
[5] L. Columbus, T. Kálai, J. Jek?, K. Hideg, W. L. Hubbell, "Molecular motion of spin labeled side chains in alpha-helices: analysis by variation of side chain structure," Biochemistry, Vol. 40, num. 13, April 2001, pp. 3828-3846. doi:10.1021/bi002645h
[6] L. Columbus, W. L. Hubbell, "A new spin on protein dynamics", Trends in Biochemical Sciences, Vol. 27, num. 6, June 2002, pp. 288-295. doi:10.1016/S0968-0004(02)02095-9
[7] G. E. Fanucci, D. S. Cafiso, "Recent advances and applications of site-directed spin labeling," Current Opinions in Structural Biology, Vol. 16, num. 5, October 2006, pp. 644-653. doi:10.1016/j.sbi.2006.08.008
[8] W. L. Hubbell, D. S. Cafiso, C. Altenbach, "Identifying conformational changes with site-directed spin labeling," Nature Structural Biology, Vol. 7, num. 9, September 2000, pp. 735-739. doi:10.1038/78956
[9] W. L. Hubbell, A. Gross, R. Langen, M. A. Lietzow, "Recent advances in site-directed spin labeling of proteins," Current Opinion in Structural Biology, Vol. 8, num. 5, October 1998, pp. 649-656. doi:10.1016/S0959-440X(98)80158-9
[10] H. S. McHaourab, M. A. Lietzow, K. Hideg, W. L. Hubbell, "Motion of spin-labeled side chains in T4 lysozyme. Correlation with protein structure and dynamics," Biochemistry, Vol. 35, num. 24, June 1996, pp. 7692-7704. doi:10.1021/bi960482k
[11] R. Noble, HIV types, subtypes, groups, and strains, Vol. 2010, AVERT, 2009.
[12] R. Kantor, R. W. Shafer, D. Katzenstein, "The HIV-1 Non-subtype B Workgroup: an international collaboration for the collection and analysis of HIV-1 non-subtype B data," Journal of the International AIDS Society, Vol. 7, num. 71, February 2005, pp. 1-3.
[13] A. Wlodawer, J. Vondrasek, "Inhibitors of HIV-1 protease: A major success of structure-assisted drug design," Annual Review of Biophysics and Biomolecular Structure, Vol. 27, 1998, pp. 249-284. doi:10.1146/annurev.biophys.27.1.249
[14] J. L. Kear, M. E. Blackburn, A. M. Veloro, B. M. Dunn, G. E. Fanucci, "Subtype polymorphisms among HIV-1 protease variants confer altered flap conformations and flexibility," Journal of the American Chemical Society, Vol. 131, num. 41, September 2009, pp. 14650-14651. doi:10.1021/ja907088a
[15] L. Galiano, M. Bonora, G. E. Fanucci, "Inter-flap distances in HIV-1 protease determined by pulsed EPR measurements," Journal of the American Chemical Society, Vol. 129, num. 36, August 2007, pp. 11004-11005. doi:10.1021/ja073684k
[16] L. Galiano, F. Ding, A. M. Veloro, M. E. Blackburn, C. Simmerling, G. E. Fanucci, "Drug pressure selected mutations in HIV-1 protease alter flap conformations," Journal of the American Chemical Society, Vol. 131, num. 2, December 2009, pp. 430-431. doi:10.1021/ja807531v
[17] M. E. Blackburn, A. M. Veloro, G. E. Fanucci, "Monitoring inhibitor induced conformational population shifts in HIV-1 protease by pulsed EPR spectroscopy," Biochemistry, Vol. 48, num. 37, September 2009, pp. 8765-8767. doi:10.1021/bi901201q
[18] A. M. Mildner, D. J. Rothrock, J. W. Leone, C. A. Bannow, J. M. Lull, I. M. Reardon, J. L. Sarcich, J. W. Howe, C. C. Tomich, C. W. Smith, R. L. Heinrikson, A. G. Tomasselli, "The HIV-1 protease as enzyme and substrate: mutagenesis of autolysis sites and generation of a stable mutant with retained kinetic properties," Biochemistry, Vol. 33, num. 32, August 1994, pp. 9405-9413. doi:10.1021/bi00198a005
[19] National Institute of Health
[20] W. Shao, L. Everitt, M. Manchester, D. D. Loeb, C. A. Hutchison, III, R. Swanstrom, "Sequence requirements of the HIV-1 protease flap region determined by saturation mutagenesis and kinetic analysis of flap mutants," Proceedings of the National Academy of Sciences USA, Vol. 94, num. 6, March 1997, pp. 2243-2248. doi:10.1073/pnas.94.6.2243
[21] L. Galiano, M. E. Blackburn, A. M. Veloro, M. Bonora, G. E. Fanucci, "Solute effects on spin labels at an aqueous-exposed site in the flap region of HIV-1 protease," Journal of Physical Chemistry B, Vol. 113, num. 6, February 2009, pp. 1673-1680. doi:10.1021/jp8057788
[22] F. Rusconi, "massXpert 2: a cross-platform software environment for polymer chemistry modelling and simulation/analysis of mass spectrometric data," Bioinformatics, Vol. 25, num. 20, 2009, pp. 2741-2742. doi:10.1093/bioinformatics/btp504
[23] J. C. Clemente, R. E. Moose, R. Hemrajani, L. R. Whitford, L. Govindasamy, R. Reutzel, R. McKenna, M. Agbandje-McKenna, M. M. Goodenow, B. M. Dunn, "Comparing the accumulation of active- and nonactive-site mutations in the HIV-1 protease," Biochemistry, Vol. 43, num. 38, September 2004, pp. 12141-12151. doi:10.1021/bi049459m
[24] B. C. Logsdon, J. F. Vickrey, P. Martin, G. Proteasa, J. I. Koepke, S. R. Terlecky, Z. Wawrzak, M. A. Winters, T. C. Merigan, L. C. Kovari, "Crystal structures of a multidrug-resistant human immunodeficiency virus type 1 protease reveal an expanded active-site cavity," Journal of Virology, Vol. 78, num. 6, March 2004, pp. 3123-3132. doi:10.1128/JVI.78.6.3123-3132.2004
[25] M. Layten, V. Hornak, C. Simmerling, "The open structure of a multi-drug-resistant HIV-1 protease is stabilized by crystal packing contacts," Journal of the American Chemical Society, Vol. 128, num. 41, October 2006, pp. 13360-13361. doi:10.1021/ja065133k
[26] P. Martin, J. F. Vickrey, G. Proteasa, Y. L. Jimenez, Z. Wawrzak, M. A. Winters, T. C. Merigan, L. C. Kovari, "Wide-open 1.3 ? structure of a multidrug-resistant HIV-1 protease as a drug target," Structure, Vol. 13, num. 12, December 2005, pp. 1887-1895. doi:10.1016/j.str.2005.11.005
[27] Z. Szeltner, L. Polgar, "Conformational stability and catalytic activity of HIV-1 protease are both enhanced at high salt concentration," Journal of Biological Chemistry, Vol. 271, num. 10, March 1996, pp. 5458-5463.
[28] V. Hornak, A. Okur, R. C. Rizzo, C. Simmerling, "HIV-1 protease flaps spontaneously open and reclose in molecular dynamics simulations," Proceedings of the National Academy of Sciences USA, Vol. 103, num. 4, January 2006, pp. 915-920. doi:10.1073/pnas.0508452103
[29] R. M. Bandaranayake, M. Prabu-Jeyabalan, J. Kakizawa, W. Sugiura, C. A. Schiffer, "Structural analysis of human immunodeficiency virus type 1 CRF01_AE protease in complex with the substrate p1-p6," Journal of Virology, Vol. 82, num. 13, July 2008, pp. 6762-6766. doi:10.1128/JVI.00018-08
[30] A. H. Robbins, R. M. Coman, E. Bracho-Sanchez, M. A. Fernandez, C. T. Gilliland, M. Li, M. Agbandje-McKenna, A. Wlodawer, B. M. Dunn, R. McKenna, "Structure of the unbound form of HIV-1 subtype A protease: comparison with unbound forms of proteases from other HIV subtypes,” Acta Crystallographica Section D: Biological Crystallography, Vol. D66, 2010, pp. 233-242. doi:10.1107/S0907444909054298
[31] R. M. Coman, A. H. Robbins, M. M. Goodenow, B. M. Dunn, R. McKenna, "High-resolution structure of unbound human immunodeficiency virus 1 subtype C protease: implications of flap dynamics and drug resistance, Acta Crystallographica Section D: Biological Crystallography, Vol. D64, 2008, pp. 754-763. doi:10.1107/S090744490801278X

  
comments powered by Disqus

Copyright © 2018 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.