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

Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 01/2011; 108(4):1331-6. DOI: 10.1073/pnas.1017877108
Source: PubMed


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°) 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.

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    • "Hydrophobic cavities, however, are not necessarily packed with hydrophobic molecules, but may also contain water wires or clusters (7,38). Empty or partially empty cavities should also make helical membrane proteins more flexible allowing them to adopt various states or sub-states (3,9,39). Taken together, hydrophobic cavities seem to be important for the stability and function of proteins, but their specific role seems to depend on the substructural context. "
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    ABSTRACT: The membrane protein packing database (MP:PD) ( is a database of helical membrane proteins featuring internal atomic packing densities, cavities and waters. Membrane proteins are not tightly packed but contain a considerable number of internal cavities that differ in volume, polarity and solvent accessibility as well as in their filling with internal water. Internal cavities are supposed to be regions of high physical compressibility. By serving as mobile hydrogen bonding donors or acceptors, internal waters likely facilitate transition between different functional states. Despite these distinct functional roles, internal cavities of helical membrane proteins are not well characterized, mainly because most internal waters are not resolved by crystal structure analysis. Here we combined various computational biophysical techniques to characterize internal cavities, reassign positions of internal waters and calculate internal packing densities of all available helical membrane protein structures and stored them in MP:PD. The database can be searched using keywords and entries can be downloaded. Each entry can be visualized in Provi, a Jmol-based protein viewer that provides an integrated display of low energy waters alongside membrane planes, internal packing density, hydrophobic cavities and hydrogen bonds.
    Nucleic Acids Research 11/2013; 42(Database issue). DOI:10.1093/nar/gkt1062 · 9.11 Impact Factor
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    • "Spectral simulations were performed using a LabVIEW (National Instruments) interface [4] of the program NLSL developed by Freed and co-workers [28], [29]. A microscopic order macroscopic disorder model was used as previously described [29]. "
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    ABSTRACT: Amyloid fibril formation is associated with a range of neurodegenerative diseases in humans, including Alzheimer's, Parkinson's, and prion diseases. In yeast, amyloid underlies several non-Mendelian phenotypes referred to as yeast prions. Mechanism of amyloid formation is critical for a complete understanding of the yeast prion phenomenon and human amyloid-related diseases. Ure2 protein is the basis of yeast prion [URE3]. The Ure2p prion domain is largely disordered. Residual structures, if any, in the disordered region may play an important role in the aggregation process. Studies of Ure2p prion domain are complicated by its high aggregation propensity, which results in a mixture of monomer and aggregates in solution. Previously we have developed a solid-support electron paramagnetic resonance (EPR) approach to address this problem and have identified a structured state for the Alzheimer's amyloid-β monomer. Here we use solid-support EPR to study the structure of Ure2p prion domain. EPR spectra of Ure2p prion domain with spin labels at every fifth residue from position 10 to position 75 show similar residue mobility profile for denaturing and native buffers after accounting for the effect of solution viscosity. These results suggest that Ure2p prion domain adopts a completely disordered structure in the native buffer. A completely disordered Ure2p prion domain implies that the amyloid formation of Ure2p, and likely other Q/N-rich yeast prion proteins, is primarily driven by inter-molecular interactions.
    PLoS ONE 10/2012; 7(10):e47248. DOI:10.1371/journal.pone.0047248 · 3.23 Impact Factor
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    • "According to the life time of the substates they often can be recognized in room temperature cw spectra of spin labeled proteins if they are characterized by different spin label side chain mobilities due to structural changes in their vicinity. In the past years, Hubbell and co-workers established three experimental techniques to analyze conformational equilibria in proteins and to dissect them from spin label rotameric exchange, namely osmolyte perturbation (Lopez et al., 2009), saturation recovery (Bridges et al., 2010) and high-pressure EPR (McCoy & Hubbell, 2011). "
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    ABSTRACT: The function of a living cell, independent of we are talking about a prokaryotic single-cellular organism or a cell in the context of an complex organism like a human, depends on intricate and balanced interaction between its components. Proteins are playing a central role in this complex cellular interaction network: Proteins interact with nucleic acids, with membranes of all cellular compartments, and, what will be in the focus of this article, with other proteins. Proteins interact to form functional units, to transmit signals for example perceived at the surface of the cell to cytoplasmic or nuclear components, or to target them to specific locations. Thus, the study of protein-protein interactions on the molecular level provides insights into the basic functional concepts of living cells and emerged as a wide field of intense research, steadily developing with the introduction of new and refined biochemical and biophysical methods. Nowadays there is a vast of methods available to study the interaction between proteins. On the biochemical level mutational studies, crosslinking experiments and chromatographic techniques provide means to identify and characterize the interfaces on the protein surface where interaction takes place. Biophysical methods include calorimetric techniques, fluorescence spectroscopy and microscopy, and "structural techniques" like X-ray crystallography, (cryo-) electron microscopy, NMR spectroscopy, FRET spectroscopy, and EPR spectroscopy on spin labelled proteins.Site-directed spin labeling (SDSL) (Altenbach et al., 1989a, 1990) in combination with electron paramagnetic resonance (EPR) spectroscopy has emerged as a powerful tool to investigate the structural and the dynamical aspects of biomolecules, under conditions close to physiological i.e. functional state of the system under exploration. The technique is applicable to soluble molecules and membrane bound proteins either solubilised in detergent or embedded in a lipid bilayer. Therein, the size and the complexity of the system under investigation is almost arbitrary (reviewed in Bordignon & Steinhoff, 2007; Hubbell et al., 1996; Hubbell et al., 1998; Klare & Steinhoff, 2009; Klug & Feix, 2007). Especially with respect to protein-protein interactions SDSL EPR can provide a vast amount of information about almost all aspects of this interaction. Spin labeling approaches can provide detailed information about the binding interface not only on the structural level but also give insights into kinetic and thermodynamic aspects of the interaction. EPR also allows determination of distances between pairs of spin labels in the range from ~ 10-80 Å with accuracies down to less than 1 Å, thereby covering a range of sized including also large multi-domain proteins and protein complexes. This chapter will give an introduction into the technique of SDSL EPR spectroscopy exemplified with data from studies on the photoreceptor/transducer-complex NpSRII/NpHtrII, followed by a number of recent examples from the literature where protein-protein interactions have been studied using this technique.
    03/2012; Intech., ISBN: 978-953-51-0244-1
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