Medical College of Wisconsin
CTSICores SearchResearch InformaticsREDCap

Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin. Proc Natl Acad Sci U S A 2013 Dec 03;110(49):E4714-22 PMID: 24248390 PMCID: PMC3856799


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.

Author List

Lerch MT, Horwitz J, McCoy J, Hubbell WL


Michael Lerch PhD Assistant Professor in the Biophysics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Circular Dichroism
Cloning, Molecular
Electron Spin Resonance Spectroscopy
Hydrogen-Ion Concentration
Models, Molecular
Protein Conformation
Spectrophotometry, Ultraviolet
Spin Labels

View this publication's entry at the Pubmed website PMID: 24248390
jenkins-FCD Prod-130 96200611f8481f0aa4f84230b11dd74d063847a3