Revisiting volume changes in pressure-induced protein unfolding

Royer, Catherine A.

doi:10.1016/s0167-4838(01)00344-2

Cited by 388 publications

(389 citation statements)

References 75 publications

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“…4D and 5C) reveals a negative Δβ T in each case; the extrema occur at P ¼ ΔV o ∕Δβ T , approximately 2.9 kbar for 46R1 and approximately 3 kbar for 118R1. The magnitude and sign of Δβ T for partial and complete unfolding of proteins is still a matter of debate, even for proteins of similar size, and likely depends on individual cases (42). Nevertheless, the negative signs of Δβ T found for the N ⇌ U and N ⇌ I equilibria are in agreement with those predicted for Δβ S (adiabatic compressibility) (43), which is similar to Δβ T near room temperature (44).…”

Section: Manipulating Populations Of Protein Conformational Substatessupporting

confidence: 70%

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

McCoy

Hubbell

2011

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Manipulating Populations Of Protein Conformational Substatessupporting

confidence: 70%

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

McCoy

Hubbell

2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Pressure is known to physically force the solvent to fill cavities inside a protein matrix, thus perturbing the native conformation. In fact, negative ⌬V values such as that calculated here for FAAH have been found in the vast majority of pressure-induced dissociation and unfolding studies (21,25).…”

Section: Faah Stability Is Strongly Dependent On Its Quaternarysupporting

confidence: 54%

Closing the Gate to the Active Site

Mei

Venere

Gasperi

et al. 2007

Journal of Biological Chemistry

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Fatty acid amide hydrolase (FAAH) is a dimeric, membranebound enzyme that degrades neuromodulatory fatty acid amides and esters and is expressed in mammalian brain and peripheral tissues. The cleavage of ≈30 amino acids from each subunit creates an FAAH variant that is soluble and homogeneous in detergent-containing buffers, opening the avenue to the in vitro mechanistic and structural studies. Here we have studied the stability of FAAH as a function of guanidinium hydrochloride concentration and of hydrostatic pressure. The unfolding transition was observed to be complex and required a fitting procedure based on a three-state process with a monomeric intermediate. The first transition was characterized by dimer dissociation, with a free energy change of ≈11 kcal/mol that accounted for ≈80% of the total stabilization energy. This process was also paralleled by a large change in the solvent-accessible surface area, because of the hydration occurring both at the dimeric interface and within the monomers. As a consequence, the isolated subunits were found to be much less stable (⌬G ≈ 3 kcal/mol). The addition of methoxyarachidonyl fluorophosphonate, an irreversible inhibitor of FAAH activity, enhanced the stability of the dimer by ≈2 kcal/mol, toward denaturant-and pressure-induced unfolding. FAAH inhibition by methoxyarachidonyl fluorophosphonate also reduced the ability of the protein to bind to the membranes. These findings suggest that local conformational changes at the level of the active site might induce a tighter interaction between the subunits of FAAH, affecting the enzymatic activity and the interaction with membranes.Fatty acid amide hydrolase (FAAH) 3 is a dimeric, integral membrane protein (1) that brings about the degradation of important fatty acid neurotransmitters, such as oleamide and anandamide (2). These fatty acid amides (FAAs), collectively termed "endocannabinoids," modulate several aspects of mammalian pathophysiology, both at central and peripheral level (3). It is now widely recognized that the biological activity of FAAs depends on the "metabolic control" of their endogenous tone (3), and in the last few years evidence has accumulated that points to FAAH as the key regulator of FAAs concentration in vivo (4, 5). Therefore, this enzyme is becoming the subject of intense investigation, and the design of ad hoc inhibitors able to tune its catalytic activity is a hot spot in medicinal chemistry (for a recent review see Ref. 6). In fact, FAAH inhibitors might represent next-generation therapeutics for the treatment of drug and alcohol abuse (7), anxiety (8), cancer (9), as well as neurodegenerative (10, 11) and vascular (12) diseases. In addition, interest toward FAAH was boosted by the fact that this enzyme is the first and, to date, the only characterized mammalian member of a large group, referred to as the "amidase signature" (AS) family (13). More than 100 members of this class have been reported in the literature, mostly from bacteria or fungi. Despite its quite remarkable size, struct...

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“…Biopolymers such as proteins remain stable and functional only in a limited pressure and temperature range [31,32,33]. Increasing temperature lead to structural changes differing from the native folded state.…”

Section: Introductionmentioning

confidence: 99%

Low-temperature and high-pressure induced swelling of a hydrophobic polymer-chain in aqueous solution

Paschek

Nonn

Geiger

2005

Phys. Chem. Chem. Phys.

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We report molecular dynamics simulations of a hydrophobic polymer-chain in aqueous solution between 260 K and 420 K at pressures of 1 bar, 3000 bar, and 4500 bar. The simulations reveal a hydrophobically collapsed state at low pressures and high temperatures. At 3000 bar and about 260 K and at 4500 bar and about 260 K, however, a transition to a swelled state is observed. The transition is driven by a smaller volume and a remarkably strong lower enthalpy of the swelled state, indicating a steep positive slope of the corresponding transition line. The swelling is stabilized almost completely by the energetically favorable state of water in the polymers hydrophobic first hydration shell at low temperatures. Although surprising, this finding is consistent with the observation of a positive heat capacity of hydrophobic solvation. Moreover, the slope and location of the observed swelling transition for the collapsed hydrophobic chain coincides remarkably well with the cold denaturation transition of proteins.

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Revisiting volume changes in pressure-induced protein unfolding

Cited by 388 publications

References 75 publications

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

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

Closing the Gate to the Active Site

Low-temperature and high-pressure induced swelling of a hydrophobic polymer-chain in aqueous solution

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