title: Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin.
abstract: Excited states of proteins may play important roles in function, yet are difficult to study spectroscopically because of their sparse population. High hydrostatic pressure increases the equilibrium population of excited states, enabling their characterization [Akasaka K (2003) Biochemistry 42:10875-85]. High-pressure site-directed spin-labeling EPR (SDSL-EPR) was developed recently to map the site-specific structure and dynamics of excited states populated by pressure. To monitor global secondary structure content by circular dichroism (CD) at high pressure, a modified optical cell using a custom MgF2 window with a reduced aperture is introduced. Here, a combination of SDSL-EPR and CD is used to map reversible structural transitions in holomyoglobin and apomyoglobin (apoMb) as a function of applied pressure up to 2 kbar. CD shows that the high-pressure excited state of apoMb at pH 6 has helical content identical to that of native apoMb, but reversible changes reflecting the appearance of a conformational ensemble are observed by SDSL-EPR, suggesting a helical topology that fluctuates slowly on the EPR time scale. Although the high-pressure state of apoMb at pH 6 has been referred to as a molten globule, the data presented here reveal significant differences from the well-characterized pH 4.1 molten globule of apoMb. Pressure-populated states of both holomyoglobin and apoMb at pH 4.1 have significantly less helical structure, and for the latter, that may correspond to a transient folding intermediate.
authors.affiliation: Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Lerch
authors.forename: Joseph
authors.initials: J
authors.lastname: Horwitz
authors.forename: John
authors.initials: J
authors.lastname: McCoy
authors.forename: Wayne L
authors.initials: WL
authors.lastname: Hubbell
mesh_terms: D002942:Circular Dichroism / Q000379:methods; D003001:Cloning, Molecular; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009211:Myoglobin / Q000737:chemistry*; D011312:Pressure; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013113:Spin Labels; D013816:Thermodynamics
chemical_list: D009211:Myoglobin; D013113:Spin Labels