Subdomain interactions as a determinant in the folding and stability of T4 lysozyme

Llinás, Manuel; Marqusee, Susan

doi:10.1002/pro.5560070110

Cited by 62 publications

(110 citation statements)

References 43 publications

(51 reference statements)

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“…The much smaller ΔV o compared to that of 118R1 suggests that the equilibrium observed is not that for the N ⇌ U equilibrium, but rather an N ⇌ I equilibrium, where I is an intermediate with incomplete unfolding. It is known that the stability of the N subdomain is lower than that for the C subdomain (38) and that intermediate folding states (I) exist for T4L in which the N and C terminal subdomains are unfolded and folded, respectively (30,33,38,39). The broad transition region of the CD-detected urea denaturation curve of 46R1 also suggests a partially unfolded intermediate state (SI Text).…”

Section: Urea] >5 M (Si Text) the Mutant Is Destabilizedmentioning

confidence: 99%

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: Urea] >5 M (Si Text) the Mutant Is Destabilizedmentioning

confidence: 99%

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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“…All genes were cloned into high copy plasmids under a T7 promoter (pAED4 or pET27). The proteins were purified using their respective published protocols with the difference that 1 mM DTT was present during all purification steps (Dabora and Marqusee 1994;Llinas and Marqusee 1998;Robic et al 2002).…”

Section: Protein Preparationmentioning

confidence: 99%

Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers

et al. 2008

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Here we report on a method that extends the study of the mechanical behavior of single proteins to the low force regime of optical tweezers. This experimental approach relies on the use of DNA handles to specifically attach the protein to polystyrene beads and minimize the non-specific interactions between the tethering surfaces. The handles can be attached to any exposed pair of cysteine residues. Handles of different lengths were employed to mechanically manipulate both monomeric and polymeric proteins. The low spring constant of the optical tweezers enabled us to monitor directly refolding events and fluctuations between different molecular structures in quasiequilibrium conditions. This approach, which has already yielded important results on the refolding process of the protein RNase H (Cecconi et al. in Science 309: 2057-2060-2005, appears robust and widely applicable to any protein engineered to contain a pair of reactive cysteine residues. It represents a new strategy to study protein folding at the single molecule level, and should be applicable to a range of problems requiring tethering of protein molecules.

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“…The folding of WT T 4 lysozyme has been examined in detail by using hydrogen-deuterium exchange approaches and has been shown to contain two domains that fold cooperatively and with distinct free energy profiles (29)(30)(31)(32). The N-terminal domain is ∼6 kcal/mol less stable Significance Pressure unfolding of proteins is a fundamental aspect of their thermodynamic response, the origins of which remain controversial.…”

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confidence: 99%

Role of cavities and hydration in the pressure unfolding of T₄lysozyme

Nucci

Fuglestad

Athanasoula

et al. 2014

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

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It is well known that high hydrostatic pressures can induce the unfolding of proteins. The physical underpinnings of this phenomenon have been investigated extensively but remain controversial. Changes in solvation energetics have been commonly proposed as a driving force for pressure-induced unfolding. Recently, the elimination of void volumes in the native folded state has been argued to be the principal determinant. Here we use the cavity-containing L99A mutant of T 4 lysozyme to examine the pressure-induced destabilization of this multidomain protein by using solution NMR spectroscopy. The cavity-containing C-terminal domain completely unfolds at moderate pressures, whereas the N-terminal domain remains largely structured to pressures as high as 2.5 kbar. The sensitivity to pressure is suppressed by the binding of benzene to the hydrophobic cavity. These results contrast to the pseudo-WT protein, which has a residual cavity volume very similar to that of the L99A-benzene complex but shows extensive subglobal reorganizations with pressure. Encapsulation of the L99A mutant in the aqueous nanoscale core of a reverse micelle is used to examine the hydration of the hydrophobic cavity. The confined space effect of encapsulation suppresses the pressure-induced unfolding transition and allows observation of the filling of the cavity with water at elevated pressures. This indicates that hydration of the hydrophobic cavity is more energetically unfavorable than global unfolding. Overall, these observations point to a range of cooperativity and energetics within the T 4 lysozyme molecule and illuminate the fact that small changes in physical parameters can significantly alter the pressure sensitivity of proteins.protein stability | protein folding and cooperativity | protein hydration | high-pressure NMR | reverse micelle encapsulation

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Subdomain interactions as a determinant in the folding and stability of T4 lysozyme

Cited by 62 publications

References 43 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

Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers

Role of cavities and hydration in the pressure unfolding of T₄lysozyme

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Subdomain interactions as a determinant in the folding and stability of T4 lysozyme

Cited by 62 publications

References 43 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

Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers

Role of cavities and hydration in the pressure unfolding of T4lysozyme

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Role of cavities and hydration in the pressure unfolding of T₄lysozyme