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 MgF 2 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.P roteins in solution are dynamic molecules, exhibiting conformational flexibility across a range of time and length scales (1). In addition to a well-ordered native state, conformational excursions to low-lying "excited states" may be required in protein function (2). For example, on a funnel-shaped energy landscape (3, 4), excited states have increased configurational entropy that may give rise to the promiscuous protein-protein interactions that define a protein interactome (5, 6). Structural changes involved in formation of the excited state may take a variety of forms, from rigid body motions of helices (7) to local unfolding of secondary structural elements (8). Despite their functional relevance, excited states are sparsely populated and may escape detection by standard spectroscopic techniques. Hydrostatic pressure apparently offers a solution to this problem by reversibly populating excited states of proteins, allowing for spectroscopic characterization (9-14). Pressure application is particularly useful because it shifts the relative population of preexisting conformational states while minimally affecting the conformational landscape itself (15).Assuming that pressure can populate excited states for study, the increased configurational entropy of such states presents a challenge to spectroscopic methods that aim to describe structure; depending on the intrinsic time scale of the method, either a population-weighted average structure or a heterogeneous ensemble is observed. The intrinsic time scale of con...

Structure-relaxation mechanism for the response of T4 lysozyme cavity mutants to hydrostatic pressure

“…In the WT* background, application of pressure to 2 kbar results in a slight reduction in nanosecond nitroxide motion as evidenced by a minor line broadening in the spectra of R1 at each site. Such effects have been previously reported and interpreted to reflect a limited compressibility of the protein in the region of the label site (30,39). In the L99A background, the pressure-dependent changes are of substantially larger magnitude.…”

“…In this model, voids are eliminated by pressure owing to an increase in the population of an alternative packing arrangement of the core in which cavities are filled with native side chains rather than solvent. This model has been suggested to play a role in certain proteins at high pressure (1,(29)(30)(31), although to our knowledge direct observation of structure relaxation under pressure has not been reported.…”

“…The experimental approach is based on site-directed spin labeling EPR (SDSL-EPR) and the recently developed technologies of high-pressure continuous wave (CW) EPR spectroscopy for SDSL (30,39), pressure-resolved double electron-electron resonance (PR DEER) spectroscopy (40), and high-pressure circular dichroism (HP-CD) (30). HP-CD reveals the global secondary structure of the protein at pressures up to 2.4 kbar.…”

“…HP-CD reveals the global secondary structure of the protein at pressures up to 2.4 kbar. CW EPR line shapes of spin-labeled proteins are sensitive to backbone fluctuations on the nanosecond time scale (41,42) and can unambiguously identify site-specific unfolding under pressure to 4 kbar, as well as identify sequences in slow conformational exchange on the microsecond-millisecond time scale (30,39). The intrinsic time scale of SDSL-EPR is much shorter than NMR, such that spectral averaging of microsecondmillisecond protein conformational exchange does not occur.…”

“…Recent development of a modified high-pressure optical cell suitable for use in far-UV CD allowed for monitoring global secondary structure in the range of 0-2.4 kbar (gauge pressure; 0 bar is equal to atmospheric pressure) (30). The pressure dependence for L99A and L99A/G113A/R119P was investigated to assess whether pressure affects the global secondary structure.…”