High-pressure EPR reveals conformational equilibria and volumetric properties of spin-labeled proteins

Abstract

Identifying equilibrium conformational exchange and characterizing conformational substates is essential for elucidating mechanisms of function in proteins. Site-directed spin labeling has previously been employed to detect conformational changes triggered by some event, but verifying conformational exchange at equilibrium is more challenging. Conformational exchange (microsecond-millisecond) is slow on the EPR time scale, and this proves to be an advantage in directly revealing the presence of multiple substates as distinguishable components in the EPR spectrum, allowing the direct determination of equilibrium constants and free energy differences. However, rotameric exchange of the spin label side chain can also give rise to multiple components in the EPR spectrum. Using spin-labeled mutants of T4 lysozyme, it is shown that high-pressure EPR can be used to: (i) demonstrate equilibrium between spectrally resolved states, (ii) aid in distinguishing conformational from rotameric exchange as the origin of the resolved states, and (iii) determine the relative partial molar volume (ΔV o ) and isothermal compressibility (Δβ T ) of conformational substates in two-component equilibria from the pressure dependence of the equilibrium constant. These volumetric properties provide insight into the structure of the substates. Finally, the pressure dependence of internal side-chain motion is interpreted in terms of volume fluctuations on the nanosecond time scale, the magnitude of which may reflect local backbone flexibility.P roteins undergo structural fluctuations that span a wide range of time scales. Among these motions are fast backbone fluctuations on the picosecond-nanosecond time scale and slower conformational fluctuations on the microsecond and longer time scale (1-3). Molecular flexibility on these time scales plays a central role in protein function (4). For example, in recognitionbinding sequences, dynamic disorder on the nanosecond-microsecond time scale may increase the rate of protein-protein interactions via a "fly casting" mechanism (5). An emerging disorder-to-order paradigm for interaction (6) can also give rise to promiscuity in binding that increases the size of the "interactome."Regulation of protein function is often linked to a conformational switch triggered by an interaction with a chemical or physical signal. One mechanistic interpretation of this event is provided by a "preequilibrium" model, which posits that all possible conformations of a protein exist at equilibrium with populations proportional to their relative energies (7). The exchange ("hopping") event between different conformers is characterized by lifetimes in the microsecond-millisecond range (1, 2, 8). In this model, a conformational switch is viewed as a shift in the relative populations of existing conformational states rather than the creation of a new state.To evaluate the above models and elucidate molecular mechanisms of protein function, it is essential to have experimental means for identifying dynamically disordered sequenc...

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

“…Consistent with HP-CD data, the CW spectra in L99A unequivocally show that the C-terminal domain is not globally unfolded at 2 kbar; in that case the CW resonance line shape would consist of sharp resonance lines rather than the reversible appearance of components corresponding to immobile states of R1 (Fig. S1C) (30,39). As shown below, the new conformational substates sensed by R1 in L99A at 2 kbar are not related to the E conformation, but likely reflect low-amplitude structural fluctuations within the ground state ensemble of L99A (but see Discussion).…”

“…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.…”

“…CW EPR spectra of spin-labeled proteins were recorded at X-band in a Bruker ELEXSYS 580 spectrometer at room temperature in buffer at pH 5.5 (50 mM MES and 25 mM NaCl) or pH 6.8 (50 mM MOPS and 25 mM NaCl) containing 30% (wt/wt) sucrose or 25% (wt/wt) Ficoll-70 (Sigma). These concentrations of sucrose and Ficoll-70 cause an equivalent increase in the effective viscosity of the solution, and are thus equally effective in reducing the contribution of rotational diffusion of the protein to the spectral lineshape (39,59,60). Protein concentrations were in the range of 100-500 μM.…”

“…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.…”

“…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.…”