On the Origin of Residual Dipolar Couplings from Denatured Proteins

Louhivuori, Martti; Pääkkönen, Kimmo; Fredriksson, Kai; Permi, Perttu; Lounila, Juhani; Annila, Arto

doi:10.1021/ja035427v

Cited by 112 publications

(128 citation statements)

References 28 publications

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“…By using residual dipolar couplings (RDCs) from NMR, Shortle and Ackerman (27) showed that native-like topology persists under strongly denaturing conditions in a truncated staphylococcal nuclease. Contention about the origin of RDCs in unfolded proteins notwithstanding (28), other NMR methods also detect structure in the unfolded state. By using triple-resonance NMR, native-like topology has been observed in protein L (29).…”

Section: Discussionmentioning

confidence: 99%

Reassessing random-coil statistics in unfolded proteins

Fitzkee

Rose

2004

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

316

325

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The Gaussian-distributed random coil has been the dominant model for denatured proteins since the 1950s, and it has long been interpreted to mean that proteins are featureless, statistical coils in 6 M guanidinium chloride. Here, we demonstrate that random-coil statistics are not a unique signature of featureless polymers. The random-coil model does predict the experimentally determined coil dimensions of denatured proteins successfully. Yet, other equally convincing experiments have shown that denatured proteins are biased toward specific conformations, in apparent conflict with the random-coil model. We seek to resolve this paradox by introducing a contrived counterexample in which largely native protein ensembles nevertheless exhibit random-coil characteristics. Specifically, proteins of known structure were used to generate disordered conformers by varying backbone torsion angles at random for Ϸ8% of the residues; the remaining Ϸ92% of the residues remained fixed in their native conformation. Ensembles of these disordered structures were generated for 33 proteins by using a torsion-angle Monte Carlo algorithm with hard-sphere sterics; bulk statistics were then calculated for each ensemble. Despite this extreme degree of imposed internal structure, these ensembles have end-to-end distances and mean radii of gyration that agree well with random-coil expectations in all but two cases.T he protein folding reaction, unfolded (U)^native (N), is a reversible disorder^order transition. Typically, proteins are disordered (U) at high temperature, high pressure, extremes of pH, or in the presence of denaturing solvents, but they fold to uniquely ordered, biologically relevant conformers (N) under physiological conditions. With some exceptions (1), the folded state is the biologically relevant form, and it can be characterized to atomic detail by using x-ray crystallography and NMR spectroscopy. In contrast, our understanding of the unfolded state is based primarily on a statistical model, the random-coil model, which was developed largely by Flory (2) and corroborated by Tanford (3) in the 1950s and 1960s.In a random coil, the energy differences among sterically accessible backbone conformers are of order ϷkT (where k is Boltzmann's constant, and T is the absolute temperature). Consequently, there are no strongly preferred conformations, the energy landscape is essentially featureless, and a Boltzmann-weighted ensemble of such polymers would populate this landscape uniformly.Our motivation here is to dispel the belief, which is widespread among protein chemists, that the presence of random-coil statistics for denatured proteins confirms the absence of residual structure in these molecules. Indeed, it is well known to polymer chemists that rods of any stiffness (e.g., steel I-beams) behave as Gaussiandistributed, temperature-dependent random coils if they are long enough. Chains in which the persistence length exceeds one physical link can be treated effectively by rewriting them as polymers of Kuhn segments (ref. 2, pa...

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Section: Discussionmentioning

confidence: 99%

Reassessing random-coil statistics in unfolded proteins

Fitzkee

Rose

2004

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

316

325

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“…We investigate whether a correlation exists between the magnitude of the RDCs and the AR of the individual members in the ensemble of unfolded state proteins. High ARs are expected for individual chains in any random-walk model even though the ensemble average is spherical (33,44). The AR is defined as (I z )͞ ͌ I x I y , where I x , I y , and I z are the moments of inertia for the given conformation in ascending order.…”

Section: Radius Of Gyrationmentioning

confidence: 99%

“…Shortle and coworkers (1,(26)(27)(28) argue that denatured proteins retain some native-like topology in the denatured state. However, others propose simpler explanations related to the fundamental nature of random coils in aligned media (33) or to short stretches of extended conformations (29,34).…”

mentioning

confidence: 99%

Statistical coil model of the unfolded state: Resolving the reconciliation problem

Jha

Colubri

Freed

et al. 2005

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

303

415

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An unfolded state ensemble is generated by using a self-avoiding statistical coil model that is based on backbone conformational frequencies in a coil library, a subset of the Protein Data Bank. The model reproduces two apparently contradicting behaviors observed in the chemically denatured state for a variety of proteins, random coil scaling of the radius of gyration and the presence of significant amounts of local backbone structure (NMR residual dipolar couplings). The most stretched members of our unfolded ensemble dominate the residual dipolar coupling signal, whereas the uniformity of the sign of the couplings follows from the preponderance of polyproline II and ␤ conformers in the coil library. Agreement with the NMR data substantially improves when the backbone conformational preferences include correlations arising from the chemical and conformational identity of neighboring residues. Although the unfolded ensembles match the experimental observables, they do not display evidence of nativelike topology. By providing an accurate representation of the unfolded state, our statistical coil model can be used to improve thermodynamic and kinetic modeling of protein folding. denatured state ͉ protein folding ͉ residual dipolar coupling ͉ nearest neighbor ͉ radius of gyration D enatured proteins are the initial state for mechanistic and thermodynamic studies of folding. The recent resurgence of interest in the unfolded state is partly motivated by the development of NMR methods that are capable of providing site-resolved structural information (1-7). These measurements indicate that unfolded proteins have far richer structural diversity than earlier believed, possibly even encoding the native topology (1,(8)(9)(10)(11).These works seem at odds with the classic studies by Tanford et al. (12,13), who demonstrated by using hydrodynamic methods that the global dimensions of denatured proteins exhibit the size dependence expected for self-avoiding ''random coil'' polymers. More recent measurements of the radius of gyration, R g , using small-angle scattering methods exhibit the same random coil scaling behavior with length R g ϰ N 0.585 (8,14). These observations are consistent with denatured proteins being random coils in good solvent conditions (15). This finding leads to the so-called ''reconciliation problem'' between the random coil scaling behavior and the presence of significant amounts of local structure in unfolded state (14, 16).However, Rose and Fitzkee (17) demonstrate that even a ''deliberately extreme'' model of chains composed of native-like segments connected by flexible residues also can reproduce random coil scaling behavior. Hence, the recapitulation of the scaling behavior provides only a weak test for any unfolded state model. Nevertheless, spectroscopic measurements, such as circular dichroism, indicate that most unfolded states, particularly chemicaldenatured proteins (8,13,18), have little secondary structure. Accordingly, the unrealistic native-like segment model is ruled out. More exacting...

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“…42 For statistical random coils, RDCs are predicted to have a characteristic profile, uniform throughout the polypeptide except at the termini, where they approach zero due to increasingly isotropic flexibility. 48 Deviations from such a profile may be an indication of secondary structure preferences with a decrease of RDC amplitude correlated with local compactness or flexibility. 49 For aS, it has also been suggested that large amplitude RDCs in the C-terminal tail of the protein may report on long-range transient contacts between the C-terminus and regions in the N-terminal lipid-binding domain.…”

Section: Residual Dipolar Couplingsmentioning

confidence: 99%

Charge neutralization and collapse of the C‐terminal tail of alpha‐synuclein at low pH

2009

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Alpha-synuclein (aS) is the primary component of Lewy bodies, the pathological hallmark of Parkinson's Disease. Aggregation of aS is thought to proceed from a primarily disordered state with nascent secondary structure through intermediate conformations to oligomeric forms and finally to mature amyloid fibrils. Low pH conditions lead to conformational changes associated with increased aS fibril formation. Here we characterize these structural and dynamic changes using solution state NMR measurements of secondary chemical shifts, relaxation parameters, residual dipolar couplings, and paramagnetic relaxation enhancement. We find that the neutralization of negatively charged side-chains eliminates electrostatic repulsion in the C-terminal tail of aS and leads to a collapse of this region at low pH. Hydrophobic contacts between the compact C-terminal tail and the NAC (non-amyloid-b component) region are maintained and may lead to the formation of a globular domain. Transient long-range contacts between the C-terminus of the protein and regions N-terminal to the NAC region are also preserved. Thus, the release of long-range contacts does not play a role in the increased aggregation of aS at low pH, which we instead attribute to the increased hydrophobicity of the protein.

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On the Origin of Residual Dipolar Couplings from Denatured Proteins

Cited by 112 publications

References 28 publications

Reassessing random-coil statistics in unfolded proteins

Reassessing random-coil statistics in unfolded proteins

Statistical coil model of the unfolded state: Resolving the reconciliation problem

Charge neutralization and collapse of the C‐terminal tail of alpha‐synuclein at low pH

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