Cross-Validation of the Structure of a Transiently Formed and Low Populated FF Domain Folding Intermediate Determined by Relaxation Dispersion NMR and CS-Rosetta

Barette, Julia; Vėlyvis, Algirdas; Religa, Tomasz L.; Korzhnev, Dmitry M.; Kay, Lewis E.

doi:10.1021/jp209974f

Cited by 15 publications

(13 citation statements)

References 92 publications

(275 reference statements)

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“…Indeed, we previously showed that point substitution mutations as well as single-atom substitutions can be used to stabilize RNA ESs 8,16,17,74 . In addition, various forms of mutagenesis have successfully been used to stabilize ESs of proteins 30,33,34,75,76 . Here, it is critically important to measure a comprehensive set of RD data that probe various aspects of conformation.…”

Section: Discussionmentioning

confidence: 99%

Atomic structures of excited state A–T Hoogsteen base pairs in duplex DNA by combining NMR relaxation dispersion, mutagenesis, and chemical shift calculations

et al. 2018

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NMR relaxation dispersion studies indicate that in canonical duplex DNA, Watson-Crick base pairs (bps) exist in dynamic equilibrium with short-lived low abundance excited state Hoogsteen bps. N1-methylated adenine (m1A) and guanine (m1G) are naturally occurring forms of damage that stabilize Hoogsteen bps in duplex DNA. NMR dynamic ensembles of DNA duplexes with m1A-T Hoogsteen bps reveal significant changes in sugar pucker and backbone angles in and around the Hoogsteen bp, as well as kinking of the duplex towards the major groove. Whether these structural changes also occur upon forming excited state Hoogsteen bps in unmodified duplexes remains to be established because prior relaxation dispersion probes provided limited information regarding the sugar-backbone conformation. Here, we demonstrate measurements of C3′ and C4′ spin relaxation in the rotating frame (R1ρ) in uniformly 13C/15N labeled DNA as sensitive probes of the sugar-backbone conformation in DNA excited states. The chemical shifts, combined with structure-based predictions using an Automated Fragmentation Quantum Mechanics/Molecular Mechanics (AFQM/MM) method, show that the dynamic ensemble of DNA duplexes containing m1A-T Hoogsteen bps accurately model the excited state Hoogsteen conformation in two different sequence contexts. Formation of excited state A-T Hoogsteen bps is accompanied by changes in sugar-backbone conformation that allow the flipped syn adenine to form hydrogen-bonds with its partner thymine and this in turn results in overall kinking of the DNA toward the major groove. Results support the assignment of Hoogsteen bps as the excited state observed in canonical duplex DNA, provide an atomic view of DNA dynamics linked to formation of Hoogsteen bps, and lay the groundwork for a potentially general strategy for solving structures of nucleic acid excited states.

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Section: Discussionmentioning

confidence: 99%

Atomic structures of excited state A–T Hoogsteen base pairs in duplex DNA by combining NMR relaxation dispersion, mutagenesis, and chemical shift calculations

et al. 2018

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“…In this manner, I becomes the dominant conformation in solution and is now "visible" using standard NMR methods. The structure of the truncated mutant was determined using classical NMR approaches and shown to be the same as that of the invisible I state elucidated as described above (38). The importance of cross-validating structures of excited states via traditional NMR studies of designed, stabilizing mutants cannot be overemphasized, especially given that the development of the RD methodology is still progressing.…”

Section: Nmr Methods For Studying Excited Biomolecular Conformationsmentioning

confidence: 99%

NMR paves the way for atomic level descriptions of sparsely populated, transiently formed biomolecular conformers

Sekhar

Kay

2013

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

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The importance of dynamics to biomolecular function is becoming increasingly clear. A description of the structure-function relationship must, therefore, include the role of motion, requiring a shift in paradigm from focus on a single static 3D picture to one where a given biomolecule is considered in terms of an ensemble of interconverting conformers, each with potentially diverse activities. In this Perspective, we describe how recent developments in solution NMR spectroscopy facilitate atomic resolution studies of sparsely populated, transiently formed biomolecular conformations that exchange with the native state. Examples of how this methodology is applied to protein folding and misfolding, ligand binding, and molecular recognition are provided as a means of illustrating both the power of the new techniques and the significant roles that conformationally excited protein states play in biology.energy landscape | transiently and sparsely populated biomolecular states | invisible states | structure-function paradigmThe field of structural biology emerged from seminal X-ray diffraction studies of biomolecules such as DNA (1), myoglobin (2), hemoglobin (3), and lysozyme (4) that were carried out over 50 years ago. Since these landmark investigations, our knowledge of the relation between structure and function has greatly expanded, driven by significant improvements in both experimental and computational approaches and, of course, by the exponential increase in the number of structures that are now available. Despite tremendous advances, structural biology remains largely focused on studies of the lowest-energy conformational states of biomolecules, and although there are notable exceptions (5), the end game often remains the determination of a single static 3D structure that is then used as a starting point for understanding molecular function. It is becoming increasingly clear, however, that this narrow approach is not sufficient and that a description of molecular structure must take into account conformational fluctuations that can occur over a broad range of both time and length scales.The conformational space that can be explored by a given biomolecule is usually explained by invoking the concept of an energy landscape (6), a multidimensional hypersurface that governs the thermodynamics and kinetics of conformational transitions (7,8). Because biomolecular stability is dictated by the sum of a large number of attractive and repulsive interactions of similar strength, the resultant free-energy landscape is rugged (9), with the global minimum in the surface thought to correspond to the native state of the biomolecule. In addition, there are often local minima that are separated from the global minimum by free-energy barriers that can be overcome by thermal fluctuations. These low-lying conformationally excited states have remained elusive to quantitative investigation because their sparse population and transient nature complicates their study by conventional structural biology techniques that are so powerfu...

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“…For the first three variants (FF 1-60 , FF , and FF 9-60 ), 1 H N -15 N HSQC spectra are very similar, with two sets of signals corresponding to states M and D of the protein, while only a single set of signals is observed in NMR spectra of FF 11-60 that belong to the monomeric form. We have previously determined the structure of FF 11-60 using a standard NOE-based approach, establishing that it is a very good structural mimic of the folding intermediate of the full-length domain (22). The finding that truncation of the N-terminal residues of the FF domain, and specifically elimination of Thr10 and Trp11, leads to a shift in the monomer-dimer equilibrium toward the monomer form suggests that these residues are responsible for stabilization of the dimer.…”

Section: Resultsmentioning

confidence: 99%

“…The plasmid-encoding FF 1-60 (residues 1-60) was obtained using the QuikChange protocol (Stratagene) by introducing a stop codon after position 60 in the gene encoding the full-length domain cloned into a modified pRSET vector (13). The plasmids for FF domain variants with truncated N termini, FF 7-60 , FF 9-60 , and FF 11-60 , were produced from a plasmid encoding FF 1-60 as described elsewhere (22). All NMR experiments were performed on samples with protein concentrations of approximately 0.3 mM, 50 mM sodium acetate, 100 mM NaCl, pH ¼ 4.9, 25°C.…”

Section: Methodsmentioning

confidence: 99%

“…In one such application they can be used to cross-validate RD NMR-derived structural models of the intermediate state, a topic of particular interest at present because RD-derived structures are only beginning to emerge. Notably, high-resolution NMR studies of a truncated variant, FF , that mimics the folding intermediate of the full-length domain have established the accuracy of the RD-based model (22). However, beyond cross-validation of This article is a PNAS Direct Submission.…”

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confidence: 99%

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Transiently populated intermediate functions as a branching point of the FF domain folding pathway

Korzhnev

Religa

Kay

2012

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

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Studies of protein folding and the intermediates that are formed along the folding pathway provide valuable insights into the process by which an unfolded ensemble forms a functional native conformation. However, because intermediates on folding pathways can serve as initiation points of aggregation (implicated in a number of diseases), their characterization assumes an even greater importance. Establishing the role of such intermediates in folding, misfolding, and aggregation remains a major challenge due to their often low populations and short lifetimes. We recently used NMR relaxation dispersion methods and computational techniques to determine an atomic resolution structure of the folding intermediate of a small protein module-the FF domain-with an equilibrium population of 2-3% and a millisecond lifetime, 25°C. Based on this structure a variant FF domain has been designed in which the native state is selectively destabilized by removing the carboxyl-terminal helix in the native structure to produce a highly populated structural mimic of the intermediate state. Here, we show via solution NMR studies of the designed mimic that the mimic forms distinct conformers corresponding to monomeric and dimeric (K d ¼ 0.2 mM) forms of the protein. The conformers exchange on the seconds timescale with a monomer association rate of 1.1·10 4 M −1 s −1 and with a region responsible for dimerization localized to the amino-terminal residues of the FF domain. This study establishes the FF domain intermediate as a central player in both folding and misfolding pathways and illustrates how incomplete folding can lead to the formation of higher-order structures.excited protein states | protein folding intermediate I t is increasingly clear that protein aggregation can be initiated from locally unfolded and/or misfolded conformations that become accessible due to thermal fluctuations from the native state (1-4). Such conformers, therefore, may play critical roles in the formation of molecular assemblies implicated in aggregationrelated diseases (1-3). However, these species are often sparsely and transiently populated (4, 5), challenging their detailed characterization using conventional tools of structural biology. Their role in aggregate formation and in protein misfolding in general is, therefore, not well understood.With the development of nuclear magnetic resonance (NMR) relaxation dispersion (RD) methods (5, 6) it has become possible to study transiently formed conformers populated at a level of 0.5% or higher (excited states), provided that they interconvert on the millisecond timescale with a populated conformation that can be observed in NMR spectra (ground state). The thermodynamics and kinetics of the exchange reaction can be accessed by RD methodology. In addition, backbone 1 H, 15 N, 13 C chemical shifts (5-7), residual dipolar couplings (RDCs) (8), and residual chemical shift anisotropies (RCSAs) (9) of the excited state can be measured, providing a rich source of structural information. By combining the RD measureme...

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Cross-Validation of the Structure of a Transiently Formed and Low Populated FF Domain Folding Intermediate Determined by Relaxation Dispersion NMR and CS-Rosetta

Cited by 15 publications

References 92 publications

Atomic structures of excited state A–T Hoogsteen base pairs in duplex DNA by combining NMR relaxation dispersion, mutagenesis, and chemical shift calculations

Atomic structures of excited state A–T Hoogsteen base pairs in duplex DNA by combining NMR relaxation dispersion, mutagenesis, and chemical shift calculations

NMR paves the way for atomic level descriptions of sparsely populated, transiently formed biomolecular conformers

Transiently populated intermediate functions as a branching point of the FF domain folding pathway

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