Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

Xue, Yi; Podkorytov, Ivan S.; Rao, D. Krishna; Benjamin, Nathan; Sun, Honglei; Skrynnikov, Nikolai R.

doi:10.1002/pro.153

Cited by 52 publications

(94 citation statements)

References 113 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…PREs, generated by attachment of a spin label to a reactive cysteine in a protein, are powerful structural constraints that have been used in a large number of structural studies of both folded (22,23) and unfolded proteins (24)(25)(26)(27)(28) and to generate atomic models of molecular complexes (29,30). The r −6 dependence of the PRE on the distance between the unpaired electron of the spin label and the affected NMR probe provides long range information that extends up to distances of 25-30 Å (31), significantly larger than those obtained from nuclear Overhauser effect (NOE) measurements.…”

Section: Resultsmentioning

confidence: 99%

Hsp70 biases the folding pathways of client proteins

Sekhar

Rosenzweig

Bouvignies

et al. 2016

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

View full text Add to dashboard Cite

The 70-kDa heat shock protein (Hsp70) family of chaperones bind cognate substrates to perform a variety of different processes that are integral to cellular homeostasis. Although detailed structural information is available on the chaperone, the structural features of folding competent substrates in the bound form have not been well characterized. Here we use paramagnetic relaxation enhancement (PRE) NMR spectroscopy to probe the existence of long-range interactions in one such folding competent substrate, human telomere repeat binding factor (hTRF1), which is bound to DnaK in a globally unfolded conformation. We show that DnaK binding modifies the energy landscape of the substrate by removing long-range interactions that are otherwise present in the unbound, unfolded conformation of hTRF1. Because the unfolded state of hTRF1 is only marginally populated and transiently formed, it is inaccessible to standard NMR approaches. We therefore developed a 1 H-based CEST experiment that allows measurement of PREs in sparse states, reporting on transiently sampled conformations. Our results suggest that DnaK binding can significantly bias the folding pathway of client substrates such that secondary structure forms first, followed by the development of longer-range contacts between more distal parts of the protein.Hsp70 | protein folding | excited states | molecular chaperones | PRE T he 70-kDa heat shock protein (Hsp70) chaperone system is an important component of the cellular proteostasis machinery, serving as a central hub to channel client proteins along folding, refolding, maturation, disaggregation, and proteolytic pathways in cooperation with other chaperone assemblies such as Hsp90, Hsp104, and GroEL/ES (1-3). Central to Hsp70 function is its ATP-dependent interaction with client proteins, facilitated by Hsp40 cochaperones and nucleotide exchange factors (NEFs) (4). Hsp70 is a weak ATPase that recognizes and binds substrates at sites containing large aliphatic hydrophobic residues, Ile, Leu, and Val, flanked by positively charged amino acids such as Arg and Lys (5). Initial binding of substrate to the ATP-form of Hsp70 can occur directly, or via Hsp40, with rapid on/off kinetics that give rise to a weak overall affinity for the interaction. Subsequent ATP hydrolysis, stimulated by interactions with Hsp40 and substrate, leads to a large conformational change in the chaperone, locking the substrate in the Hsp70 bound state. The resulting complex is of high affinity with slow substrate on/off rates (2).Escherichia coli DnaK is the best studied of Hsp70 chaperones. It is a 70-kDa protein comprised of an N-terminal ATPase and a C-terminal substrate binding domain that communicate allosterically to couple ATP hydrolysis with substrate binding (3). Highresolution structures of ADP-(6) and ATP-DnaK (7, 8) establish that these two domains dock on to one another in the ATP-DnaK state, but become detached from each other in the ADP-bound form. In contrast to the detailed structural studies characterizing DnaK, little atomic...

show abstract

Section: Resultsmentioning

confidence: 99%

Hsp70 biases the folding pathways of client proteins

Sekhar

Rosenzweig

Bouvignies

et al. 2016

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

View full text Add to dashboard Cite

show abstract

“…This 'Gillespie-Shortle' model has been shown to be applicable to unfolded proteins 74,75 . For model calculations of the random flight chain, r is given by the expectation value for a random flight chain 〈 〉 r Nl 2 2 = , where N is the distance in residues from the spin label, and l is the length of an amino acid residue backbone, 3.8 Å (ref.…”

Section: Competing Financial Interestsmentioning

confidence: 99%

Conformation and dynamics of the periplasmic membrane-protein–chaperone complexes OmpX–Skp and tOmpA–Skp

Burmann

Wang

Hiller

2013

Nat Struct Mol Biol

134

241

View full text Add to dashboard Cite

The biogenesis of integral outer-membrane proteins (OMPs) in Gram-negative bacteria requires molecular chaperones that prevent the aggregation of OMP polypeptides in the aqueous periplasmic space. How these energy-independent chaperones interact with their substrates is not well understood. We have used high-resolution NMR spectroscopy to examine the conformation and dynamics of the Escherichia coli periplasmic chaperone Skp and two of its complexes with OMPs. The Skp trimer constitutes a flexible architectural scaffold that becomes more rigid upon substrate binding. The OMP substrates populate a dynamic conformational ensemble with structural interconversion rates on the submillisecond timescale. The global lifetime of the chaperone-substrate complex is seven orders of magnitude longer, emerging from the short local lifetimes by avidity. The dynamic state allows for energy-independent substrate release and provides a general paradigm for the conformation of OMP polypeptides bound to energy-independent chaperones.

show abstract

“…Nitroxide spin labels cause significant broadening of NMR resonances of spins that are within 20 Å of the spin label (40)(41)(42). These effects provide a useful probe for long-range contacts (15,19,30,43,44). Spin labels were attached at positions 2, 10, 32, 49, and 51, respectively (SI Appendix, Fig.…”

Section: Ntl9 Samples Predominantly Expanded Conformations With Neglimentioning

confidence: 99%

Experiments and simulations show how long-range contacts can form in expanded unfolded proteins with negligible secondary structure

Meng

Lyle

Luan

et al. 2013

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

111

View full text Add to dashboard Cite

The sizes of unfolded proteins under highly denaturing conditions scale as N 0.59 with chain length. This suggests that denaturing conditions mimic good solvents, whereby the preference for favorable chain-solvent interactions causes intrachain interactions to be repulsive, on average. Beyond this generic inference, the broader implications of N 0.59 scaling for quantitative descriptions of denatured state ensembles (DSEs) remain unresolved. Of particular interest is the degree to which N 0.59 scaling can simultaneously accommodate intrachain attractions and detectable long-range contacts. Here we present data showing that the DSE of the N-terminal domain of the L9 (NTL9) ribosomal protein in 8.3 M urea lacks detectable secondary structure and forms expanded conformations in accord with the expected N 0.59 scaling behavior. Paramagnetic relaxation enhancements, however, indicate the presence of detectable long-range contacts in the denatured-state ensemble of NTL9. To explain these observations we used atomistic thermal unfolding simulations to identify ensembles whose properties are consistent with all of the experimental observations, thus serving as useful proxies for the DSE of NTL9 in 8.3 M urea. Analysis of these ensembles shows that residual attractions are present under mimics of good solvent conditions, and for NTL9 they result from low-likelihood, medium/longrange contacts between hydrophobic residues. Our analysis provides a quantitative framework for the simultaneous observation of N 0.59 scaling and low-likelihood long-range contacts for the DSE of NTL9. We propose that such low-likelihood intramolecular hydrophobic clusters might be a generic feature of DSEs that play a gatekeeping role to protect against aggregation during protein folding.atomistic simulations | denatured proteins | paramagnetic relaxation Q uantitative descriptions of unfolded proteins are important for understanding collapse transitions (1), protein folding mechanisms (2), misfolding, aggregation (3, 4), and the effects of macromolecular crowding on protein stability (5, 6). Expanded unfolded states are sampled in high concentrations of chemical denaturants such as urea and guanidinium chloride. The sizes of these denatured proteins, quantified using hydrodynamic radii (〈R h 〉) or radii of gyration (〈R g 〉), scale as N 0.59 with chain length (7-9). This N 0.59 scaling arises because denatured proteins expand to make favorable contacts with the surrounding solvent, implying that high concentrations of denaturants are good solvents for generic proteins.In good solvents, the ensemble-averaged interresidue pair interaction coefficient is positive, suggesting that the preference for favorable chain-solvent interactions leads to intrachain interactions being repulsive on average (10). The validity of this inference for denatured proteins has been demonstrated recently using a combination of single-molecule experiments and polymer theory (11). Quantitative descriptions of chain statistics for polymers in good solvents rely on the so-ca...

show abstract

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

Cited by 52 publications

References 113 publications

Hsp70 biases the folding pathways of client proteins

Hsp70 biases the folding pathways of client proteins

Conformation and dynamics of the periplasmic membrane-protein–chaperone complexes OmpX–Skp and tOmpA–Skp

Experiments and simulations show how long-range contacts can form in expanded unfolded proteins with negligible secondary structure

Contact Info

Product

Resources

About