Abstract:
:Understanding the molecular mechanisms of osmolyte protection in protein stability has proved to be challenging. In particular, little is known about the role of osmolytes in the structure of the unfolding transition state of a protein, the main determinant of its dynamics. We have developed an experimental protocol to directly probe the transition state of a protein in a range of osmolyte environments. We use an atomic force microscope in force-clamp mode to apply mechanical forces to the protein I27 and obtain force-dependent rate constants of protein unfolding. We measure the distance to the unfolding transition state, Δx(u), along a 1D reaction coordinate imposed by mechanical force. We find that for the small osmolytes, ethylene glycol, propylene glycol, and glycerol, Δx(u) scales with the size of the molecule, whereas for larger osmolytes, sorbitol and sucrose, Δx(u) remains the same as that measured in water. These results are in agreement with steered molecular dynamics simulations that show that small osmolytes act as solvent bridges in the unfolding transition state structure, whereas only water molecules act as solvent bridges in large osmolyte environments. These results demonstrate that novel force protocols combined with solvent substitution can directly probe angstrom changes in unfolding transition state structure. This approach creates new opportunities to gain molecular level understanding of the action of osmolytes in biomolecular processes.
journal_name
Proc Natl Acad Sci U S Aauthors
Dougan L,Genchev GZ,Lu H,Fernandez JMdoi
10.1073/pnas.1101934108subject
Has Abstractpub_date
2011-06-14 00:00:00pages
9759-64issue
24eissn
0027-8424issn
1091-6490pii
1101934108journal_volume
108pub_type
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