Extending the Range of Microsecond-to-Millisecond Chemical Exchange Detected in Labeled and Unlabeled Nucleic Acids by Selective Carbon R<sub>1ρ</sub> NMR Spectroscopy

Hansen, Alexandar L.; Nikolova, Evgenia N.; Casiano-Negroni, Anette; Al‐Hashimi, Hashim M.

doi:10.1021/ja8091399

Cited by 113 publications

(169 citation statements)

References 21 publications

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“…These experiments are conceptually similar to CPMG RD methods, with the variation of R 1ρ as a function of rf field strength and offset providing a sensitive measure of exchange rates and, in favorable cases, also of chemical shifts. Recent applications of the R 1ρ technique have included studies of rate processes with k ex as fast as 40,000 s −1 (26)(27)(28). PRE measurements become powerful in the case of rapidly exchanging conformational states where the effective R 2 values of NMR spins are population-weighted averages of rates in the G and E states (14).…”

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.

233

202

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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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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.

233

202

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“…Techniques based on NMR relaxation dispersion (RD) 27–29 make it possible to characterize low-abundance short-lived conformational states, or ‘excited states’ (ESs), in biomolecules 30 . Using NMR RD, we recently provided evidence that wobble G•T/U mismatches exist in dynamic equilibrium with tautomeric (ES1) and anionic (ES2) WC-like mismatches within DNA and RNA duplexes 8,31 .…”

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confidence: 99%

Dynamic basis for dG•dT misincorporation via tautomerization and ionization

Kimsey

Szymanski

Zahurancik

et al. 2018

Nature

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131

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Tautomeric and anionic Watson-Crick-like mismatches play important roles in replication and translation errors through mechanisms that are not fully understood. Using NMR relaxation dispersion, we resolved a sequence-dependent kinetic network connecting G•T/U wobbles with three distinct Watson-Crick mismatches consisting of two rapidly exchanging tautomeric species (Genol•T/U⇌G•Tenol/Uenol; population <0.4%) and one anionic species (G•T−/U−; population ≈0.001% at neutral pH). Inserting the sequence-dependent tautomerization/ionization step into a minimal kinetic mechanism for correct incorporation during replication following initial nucleotide binding leads to accurate predictions of dG•dT misincorporation probability across different polymerases, pH conditions, and for a chemically modified nucleotide, and provides mechanisms for sequence-dependent misincorporation. Our results indicate that the energetic penalty for tautomerization/ionization accounts for ≈10−2−10−3–fold discrimination against misincorporation, which proceeds primarily via tautomeric dGenol•dT and dG•dTenol with contributions from anionic dG•dT− dominating at pH ≥8.4 or for some mutagenic nucleotides.

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“…Using NMR carbon R1ρ relaxation dispersion (17)(18)(19), we recently reported (16) that the apical loop of the transactivation response element (TAR) from the HIV-1 RNA (20) rapidly exchanges (k ex = k 1 + k -1 = ∼25 kHz) with an ES that has a population (p B ) of ∼13% and exceptionally short lifetime (τ) of ∼45 μs (Fig. 1A).…”

mentioning

confidence: 99%

Invisible RNA state dynamically couples distant motifs

Lee

Dethoff

Al‐Hashimi

2014

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

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113

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Using on-and off-resonance carbon and nitrogen R1ρ NMR relaxation dispersion in concert with mutagenesis and NMR chemical shift fingerprinting, we show that the transactivation response element RNA from the HIV-1 exists in dynamic equilibrium with a transient state that has a lifetime of ∼2 ms and population of ∼0.4%, which simultaneously remodels the structure of a bulge, stem, and apical loop. This is accomplished by a global change in strand register, in which bulge residues pair up with residues in the upper stem, causing a reshuffling of base pairs that propagates to the tip of apical loop, resulting in the creation of three noncanonical base pairs. Our results show that transient states can remodel distant RNA motifs and possibly give rise to mechanisms for rapid long-range communication in RNA that can be harnessed in processes such as cooperative folding and ribonucleoprotein assembly.NMR spectroscopy | dynamics | R1ρ relaxation dispersion | nucleic acids I t is now well-established that RNA sequences do not code for a single static structure, but rather, many conformations that populate energetic minima along a free-energy landscape (1, 2). Cellular inputs, ranging from changes in temperature and pH to the binding of proteins, other RNAs, and ligands, can preferentially stabilize select conformations along the landscape, resulting in dynamic changes in RNA structure that drive the multistep catalytic cycles of ribozymes (3), regulatory activities of riboswitches (4) and other RNA-based switches (5), and the dynamic assembly and disassembly of ribonucleoprotein (RNP) complexes (6).A common mode of RNA dynamics involves rearrangements in secondary structure that can melt or create entire hairpins, and thereby expose or sequester key regulatory elements that are several nucleotides long (1,4,7,8). Such secondary structural transitions entail large kinetic barriers, so they are often catalyzed by RNA-binding proteins (9), ATP-dependent chaperones (10), or otherwise occur by modulating cotranscriptional folding (5, 11). Recently, NMR R1ρ relaxation dispersion experiments (12-15) in concert with mutagenesis (16) have helped uncover more labile RNA secondary structural transitions that can take place without assistance from external cofactors at rates that are 2-4 orders of magnitude faster than larger-scale secondary structural rearrangements. These transitions entail excursions away from the energetically favorable ground state (GS) toward lowpopulated (typically populations <15%) and short-lived (lifetime < milliseconds) species often referred to as "excited states" (ES) (12, 13). These invisible RNA ES feature localized reshuffling of base pairing in and around noncanonical motifs such as bulges, internal loops, and apical loops (16) which can also expose or sequester functionally important residues or promote ATPindependent large-scale changes in secondary structure (14, 15). These faster and more localized changes in secondary structure may meet unique demands in RNA-based regulatory functions (16).Using...

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Extending the Range of Microsecond-to-Millisecond Chemical Exchange Detected in Labeled and Unlabeled Nucleic Acids by Selective Carbon R_1ρ NMR Spectroscopy

Cited by 113 publications

References 21 publications

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

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

Dynamic basis for dG•dT misincorporation via tautomerization and ionization

Invisible RNA state dynamically couples distant motifs

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Extending the Range of Microsecond-to-Millisecond Chemical Exchange Detected in Labeled and Unlabeled Nucleic Acids by Selective Carbon R1ρ NMR Spectroscopy

Cited by 113 publications

References 21 publications

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

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

Dynamic basis for dG•dT misincorporation via tautomerization and ionization

Invisible RNA state dynamically couples distant motifs

Contact Info

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Extending the Range of Microsecond-to-Millisecond Chemical Exchange Detected in Labeled and Unlabeled Nucleic Acids by Selective Carbon R_1ρ NMR Spectroscopy