NMR analysis of main-chain conformational preferences in an unfolded fibronectin-binding protein

Penkett, Christopher J.; Redfield, Christina; Dodd, Ian B.; Hubbard, Julia A.; McBay, Diane L.; Mossakowska, Danuta E.; Smith, Richard A.; Dobson, Christopher M.; Smith, Lorna J.

doi:10.1006/jmbi.1997.1369

Cited by 122 publications

(148 citation statements)

References 55 publications

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“…NMR measurements of 3 J HN␣ coupling constants reflect individual residue's backbone dihedral angles and also largely support the view that proteins are statistical coils (19)(20)(21)(22)(23). 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).…”

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confidence: 58%

“…These frequencies generate a statistical potential that accurately reproduces the known helical, ␤-sheet, and PPII propensities (25). The statistical potential can be chosen to include correlations between adjacent residues to account for both residue type and conformation, as emphasized in our previous studies (25,35) and other earlier studies of nearest neighbor (NN) effects (20,23,(36)(37)(38)(39)(40). After assigning backbone dihedral angles according to the statistical potential, unfolded chain conformations are slightly ''nudged'' to satisfy excluded volume constraints.…”

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confidence: 99%

“…Here, we generate a statistical coil model for the unfolded state (19)(20)(21)(22)(23). The backbone conformations are determined by their frequencies in regions outside of, and not adjacent to, helices, sheets, and turns in high-resolution crystal structures (25).…”

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confidence: 99%

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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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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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“…Examples include the cell-cycle kinase inhibitor p21 waf1/Cip1/Sdi1 (43), the FlgM flagellar protein of Salmonella typhimurium (44), and the fibronectin receptor-binding protein (45). The fast inactivation domain of the Shaker B K ϩ channel acts in a structure-independent manner (46,47).…”

Section: Discussionmentioning

confidence: 99%

A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution

Ostedgaard

Baldursson

Vermeer

et al. 2000

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

112

114

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Phosphorylation of the regulatory (R) domain initiates cystic fibrosis transmembrane conductance regulator (CFTR) Cl ؊ channel activity. To discover how the function of this domain is determined by its structure, we produced an R domain protein (R8) that spanned residues 708 -831 of CFTR. Phosphorylated, but not unphosphorylated, R8 stimulated activity of CFTR channels lacking this domain, indicating that R8 is functional. Unexpectedly, this functional R8 was predominantly random coil, as revealed by CD and limited proteolysis. The CD spectra of both phosphorylated and nonphosphorylated R8 were similar in aqueous buffer. The folding agent trimethylamine N-oxide induced only a small increase in the helical content of nonphosphorylated R8 and even less change in the helical content of phosphorylated R8. These data, indicating that the R domain is predominantly random coil, may explain the seemingly complex way in which phosphorylation regulates CFTR channel activity.

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“…Examples include the cell cycle kinase inhibitor p21 waf1/Cip1/Sdi1 (43), the FlgM flagellar protein of Salmonella typhimurium (44), and the fibronectin receptor-binding protein (45). Interestingly, a regulatory sequence in another channel, the fast inactivation domain of the Shaker B K ϩ channel, also acts in a structure-independent manner (33).…”

Section: Advantages Of Intrinsically Unstructured Domainsmentioning

confidence: 99%

Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Cl− Channel by Its R Domain

Ostedgaard

Baldursson

Welsh

2001

Journal of Biological Chemistry

113

110

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Since the sequence of the CFTR 1 Cl Ϫ channel was discovered, the function of its R domain has been puzzling. CFTR is a member of the ATP-binding cassette (ABC) transporter family, and it contains the features characteristic of this family: two nucleotide binding domains (NBDs) and two membrane-spanning domains (MSDs) (1). Yet in addition, CFTR also contains the R domain, a unique sequence not found in other ABC transporters or any other proteins. The R domain serves as the major physiologic regulator of the CFTR Cl Ϫ channel (2, 3). Upon elevation of cAMP levels, cAMP-dependent protein kinase (PKA) phosphorylates the R domain allowing ATP to open and close the channel. Yet how phosphorylation activates the channel is not well understood. Some models propose that the R domain prevents the channel from opening and that phosphorylation relieves this inhibition. Other models suggest that phosphorylation of the R domain stimulates activity. Here we briefly review current knowledge that may help explain the function of this interesting domain. Sequence and Boundaries of the R DomainThe R domain was originally defined as those residues encoded by exon 13 (aa 590 -830) ( Fig. 1) (1). Although the precise boundaries of the R domain remain uncertain, recent studies suggest that the N-terminal portion of exon 13 is actually part of NBD1, whereas the C-terminal portion constitutes the structural and functional "R domain." Supporting this conclusion, the N-terminal portion of exon 13 has sequence similarity with NBDs of other ABC transporters (4, 5). More recently, crystal structures were solved for the NBDs of the ABC transporters HisP, MalK, and Rad50 (6 -8). In CFTR, residues analogous to those NBDs would extend to approximately aa 642. Experimental evidence for the boundaries came with the demonstration that deletion of aa 708 -835, but not deletions extending further in the N-terminal direction, produced a channel that was processed correctly, opened in the presence of ATP, and had conductive properties like those of wild-type CFTR (4, 9). In addition, severing CFTR after residues 633 or 835 and coexpressing the two halves in Xenopus oocytes produced functional channels, whereas severing the channel between exons 12 and 13 abolished function (10, 11). All these data suggest that the N-terminal R domain boundary begins between residues 634 and 708 and that the C-terminal boundary ends in the region of residue 835.The most striking feature of the R domain is the presence of multiple consensus PKA phosphorylation sites that are highly conserved across species (Fig. 1). There is little other sequence similarity in the R domain, unlike the substantial sequence conservation throughout the rest of CFTR. We are not aware of any other conserved protein motifs in the R domain, although there is a relatively high percentage (28%) of charged residues. Sixteen of the 24 basic residues are in PKA consensus sites. Several of the acidic residues are clustered within 817-838 (12). Phosphorylation of the R DomainPKA is the primary kinas...

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NMR analysis of main-chain conformational preferences in an unfolded fibronectin-binding protein

Cited by 122 publications

References 55 publications

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

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

A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution

Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Cl− Channel by Its R Domain

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