Insights into Conformation and Dynamics of Protein GB1 During Folding and Unfolding by NMR

Ding, Kan; Louis, John M.; Gronenborn, Angela M.

doi:10.1016/j.jmb.2003.11.042

Cited by 78 publications

(84 citation statements)

References 32 publications

(21 reference statements)

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“…In particular, the loop region between residues 14 and 17, previously identified as a melting hot spot in protein G (40), and the following 4 residues of strand ␤ 2 , exhibit lower-order parameters, corresponding to additional motion that must be occurring on the slower time scale. If we examine the order parameters of sites involved in the previously discussed correlated motions across the ␤-sheet, we find that the S slow 2 values of the sites exhibiting larger ␥-motions (K9, V11, T58 and T60) are systematically lower (0.68 Ϯ 0.03) than the respective fast motion S fast 2 for these sites (0.86 Ϯ 0.01), indicating that the correlated motions traversing the ␤-sheet are indeed occurring on the slower time range extending from tens of nanoseconds to a few milliseconds.…”

Section: Alternation Of Hydrophobic Side Chains and ␤-Sheetmentioning

confidence: 99%

Identification of slow correlated motions in proteins using residual dipolar and hydrogen-bond scalar couplings

Bouvignies

Bernadó

Meier

et al. 2005

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

225

311

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Despite their importance for biological activity, slower molecular motions beyond the nanosecond range remain poorly understood. We have assembled an unprecedented set of experimental NMR data, comprising up to 27 residual dipolar couplings per amino acid, to define the nature and amplitude of backbone motion in protein G using the Gaussian axial fluctuation model in three dimensions. Slower motions occur in the loops, and in the ␤-sheet, and are absent in other regions of the molecule, including the ␣-helix. In the ␤-sheet an alternating pattern of dynamics along the peptide sequence is found to form a long-range network of slow motion in the form of a standing wave extending across the ␤-sheet, resulting in maximal conformational sampling at the interaction site. The alternating nodes along the sequence match the alternation of strongly hydrophobic side chains buried in the protein core. Confirmation of the motion is provided through extensive crossvalidation and by independent hydrogen-bond scalar coupling analysis that shows this motion to be correlated. These observations strongly suggest that dynamical information can be transmitted across hydrogen bonds and have important implications for understanding collective motions and long-range information transfer in proteins.protein dynamics ͉ slow motions ͉ correlated M olecular dynamics, manifest in backbone and side-chain mobilities, play a crucial role in protein stability and function (1-4). The accurate characterization and understanding of protein motions thus adds an additional dimension to the structural information derived from genomics projects (5, 6). Although local backbone fluctuations on the picosecond to nanosecond time scale have been the subject of detailed characterization using NMR (7, 8) and molecular dynamics simulations (2), slower motions, in the submicrosecond to second range, remain poorly understood. Relaxation dispersion has been used to successfully identify sites of conformational exchange between states experiencing different chemical shifts in peptides (9) and proteins (10), but specific geometric motional models are often difficult to extract from these data. Slow time scales are, however, of particular interest because functionally important biological processes, including enzyme catalysis (11), signal transduction (12), ligand binding, and allosteric regulation (13), as well as collective motions involving groups of atoms or whole amino acids (14), are expected to occur in this time range. Residual dipolar couplings (RDCs) report on averages over longer time scales (up to the millisecond range) and therefore encode key information for understanding slower protein motions over a very broad time scale (15,16). Recent studies have exploited the orientational averaging properties of RDCs to characterize the amplitude and direction of motions of NH vectors (17)(18)(19) or to study local variations in position and dynamics of the amide proton (20,21). Despite this activity, key questions remain concerning the nature and amplitude of ...

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Section: Alternation Of Hydrophobic Side Chains and ␤-Sheetmentioning

confidence: 99%

Identification of slow correlated motions in proteins using residual dipolar and hydrogen-bond scalar couplings

Bouvignies

Bernadó

Meier

et al. 2005

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

225

311

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“…However, with the exception of eglin, 60 no such correlations between native and denatured states have been found in other proteins. Examples include protein GB1, 61 apomyoglobin, 52 full-length staphylococcal nuclease, and a further staphylococcal nuclease truncation mutant. 62 In the latter study, RDCs of the full-length protein and the truncation mutant were uncorrelated in the folded state, which can be explained by the different alignment properties of the two proteins.…”

Section: Experimental Rdc Profiles Of Unfolded Polypeptidesmentioning

confidence: 99%

Conformational distributions of unfolded polypeptides from novel NMR techniques

Meier

Blackledge

Grzesiek

2008

The Journal of Chemical Physics

116

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How the information content of an unfolded polypeptide sequence directs a protein towards a well-formed three-dimensional structure during protein folding remains one of the fundamental questions in structural biology. Unfolded proteins have recently attracted further interest due to their surprising prevalence in the cellular milieu, where they fulfill not only central regulatory functions, but also are implicated in diseases involving protein aggregation. The understanding of both the protein folding transition and these often natively unfolded proteins hinges on a more detailed experimental characterization of the conformations and conformational transitions in the unfolded state. This description is intrinsically very difficult due to the very large size of the conformational space. In principle, solution NMR can monitor unfolded polypeptide conformations and their transitions at atomic resolution. However, traditional NMR parameters such as chemical shifts, J couplings, and nuclear Overhauser enhancements yield only rather limited and often qualitative descriptions. This situation has changed in recent years by the introduction of residual dipolar couplings and paramagnetic relaxation enhancements, which yield a high number of well-defined, quantitative parameters reporting on the averages of local conformations and long-range interactions even under strongly denaturing conditions. This information has been used to obtain plausible all-atom models of the unfolded state at increasing accuracy. Currently, the best working model is the coil model, which derives amino acid specific local conformations from the distribution of amino acid torsion angles in the nonsecondary structure conformations of the protein data bank. Deviations from the predictions of such models can often be interpreted as increased order resulting from long-range contacts within the unfolded ensemble.

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“…However, ␣ and polyproline II (PPII) conformations have similar values (24), and nearly as good agreement with experiment can be obtained by using conformational preferences based on the entire Protein Data Bank (PDB), which is dominated by ␣ conformers, than by using ones based on unstructured regions in a coil library (25), which is dominated by PPII and ␤ conformations (data not shown). Hence, these measurements likewise do not yield a stringent test for statistical coil behavior.NMR residual dipolar coupling constants (RDCs) provide a powerful site-resolved tool for probing the structure of denatured proteins (1,10,11,(26)(27)(28)(29)(30). RDCs probe the orientation of bond vectors (generally backbone amide NH) relative to an alignment tensor fixed in the molecular frame.…”

mentioning

confidence: 99%

“…NMR residual dipolar coupling constants (RDCs) provide a powerful site-resolved tool for probing the structure of denatured proteins (1,10,11,(26)(27)(28)(29)(30). RDCs probe the orientation of bond vectors (generally backbone amide NH) relative to an alignment tensor fixed in the molecular frame.…”

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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Insights into Conformation and Dynamics of Protein GB1 During Folding and Unfolding by NMR

Cited by 78 publications

References 32 publications

Identification of slow correlated motions in proteins using residual dipolar and hydrogen-bond scalar couplings

Identification of slow correlated motions in proteins using residual dipolar and hydrogen-bond scalar couplings

Conformational distributions of unfolded polypeptides from novel NMR techniques

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

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