Characterizing micro-to-millisecond chemical exchange in nucleic acids using off-resonance R1? relaxation dispersion.
ABSTRACT: This review describes off-resonance R1? relaxation dispersion NMR methods for characterizing microsecond-to-millisecond chemical exchange in uniformly 13C/15N labeled nucleic acids in solution. The review opens with a historical account of key developments that formed the basis for modern R1? techniques used to study chemical exchange in biomolecules. A vector model is then used to describe the R1? relaxation dispersion experiment, and how the exchange contribution to relaxation varies with the amplitude and frequency offset of an applied spin-locking field, as well as the population, exchange rate, and differences in chemical shifts of two exchanging species. Mathematical treatment of chemical exchange based on the Bloch-McConnell equations is then presented and used to examine relaxation dispersion profiles for more complex exchange scenarios including three-state exchange. Pulse sequences that employ selective Hartmann-Hahn cross-polarization transfers to excite individual 13C or 15N spins are then described for measuring off-resonance R1?(13C) and R1?(15N) in uniformly 13C/15N labeled DNA and RNA samples prepared using commercially available 13C/15N labeled nucleotide triphosphates. Approaches for analyzing R1? data measured at a single static magnetic field to extract a full set of exchange parameters are then presented that rely on numerical integration of the Bloch-McConnell equations or the use of algebraic expressions. Methods for determining structures of nucleic acid excited states are then reviewed that rely on mutations and chemical modifications to bias conformational equilibria, as well as structure-based approaches to calculate chemical shifts. Applications of the methodology to the study of DNA and RNA conformational dynamics are reviewed and the biological significance of the exchange processes is briefly discussed.
Project description:Exploration of dynamic processes in proteins and nucleic acids by spin-locking NMR experiments has been facilitated by the development of theoretical expressions for the R1? relaxation rate constant covering a variety of kinetic situations. Herein, we present a generalized approximation to the chemical exchange, Rex, component of R1? for arbitrary kinetic schemes, assuming the presence of a dominant major site population, derived from the negative reciprocal trace of the inverse Bloch-McConnell evolution matrix. This approximation is equivalent to first-order truncation of the characteristic polynomial derived from the Bloch-McConnell evolution matrix. For three- and four-site chemical exchange, the first-order approximations are sufficient to distinguish different kinetic schemes. We also introduce an approach to calculate R1? for linear N-site schemes, using the matrix determinant lemma to reduce the corresponding 3N×3N Bloch-McConnell evolution matrix to a 3×3 matrix. The first- and second order-expansions of the determinant of this 3×3 matrix are closely related to previously derived equations for two-site exchange. The second-order approximations for linear N-site schemes can be used to obtain more accurate approximations for non-linear N-site schemes, such as triangular three-site or star four-site topologies. The expressions presented herein provide powerful means for the estimation of Rex contributions for both low (CEST-limit) and high (R1?-limit) radiofrequency field strengths, provided that the population of one state is dominant. The general nature of the new expressions allows for consideration of complex kinetic situations in the analysis of NMR spin relaxation data.
Project description:Spin relaxation in the rotating frame (R1?) is a powerful NMR technique for characterizing fast microsecond timescale exchange processes directed toward short-lived excited states in biomolecules. At the limit of fast exchange, only k(ex)=k(1)+k(-1) and ?ex=p(G)p(E)(??)(2) can be determined from R1? data limiting the ability to characterize the structure and energetics of the excited state conformation. Here, we use simulations to examine the uncertainty with which exchange parameters can be determined for two state systems in intermediate-to-fast exchange using off-resonance R1? relaxation dispersion. R1? data computed by solving the Bloch-McConnell equations reveals small but significant asymmetry with respect to offset (R1? (??)?R1? (-??)), which is a hallmark of slow-to-intermediate exchange, even under conditions of fast exchange for free precession chemical exchange line broadening (k(ex)/??>10). A grid search analysis combined with bootstrap and Monte-Carlo based statistical approaches for estimating uncertainty in exchange parameters reveals that both the sign and magnitude of ?? can be determined at a useful level of uncertainty for systems in fast exchange (k(ex)/??<10) but that this depends on the uncertainty in the R1? data and requires a thorough examination of the multidimensional variation of ?(2) as a function of exchange parameters. Results from simulations are complemented by analysis of experimental R1? data measured in three nucleic acid systems with exchange processes occurring on the slow (k(ex)/??=0.2; pE=?0.7%), fast (k(ex)/??=?10-16; p(E)=?13%) and very fast (k(ex)=39,000 s(-1)) chemical shift timescales.
Project description:PURPOSE:CEST is commonly used to probe the effects of chemical exchange. Although R1? asymmetry quantification has also been described as a promising option for detecting the effects of chemical exchanges, the existing acquisition approaches are highly susceptible to B1 RF and B0 field inhomogeneities. To address this problem, we report a new R1? asymmetry imaging approach, AC-iTIP, which is based on the previously reported techniques of irradiation with toggling inversion preparation (iTIP) and adiabatic continuous wave constant amplitude spin-lock RF pulses (ACCSL). We also derived the optimal spin-lock RF pulse B1 amplitude that yielded the greatest R1? asymmetry. METHODS:Bloch-McConnell simulations were used to verify the analytical formula derived for the optimal spin-lock RF pulse B1 amplitude. The performance of the AC-iTIP approach was compared to that of the iTIP approach based on hard RF pulses and the R1? -spectrum acquired using adiabatic RF pulses with the conventional fitting method. Comparisons were performed using Bloch-McConnell simulations, phantom, and in vivo experiments at 3.0T. RESULTS:The analytical prediction of the optimal B1 was validated. Compared to the other 2 approaches, the AC-iTIP approach was more robust under the influences of B1 RF and B0 field inhomogeneities. A linear relationship was observed between the measured R1? asymmetry and the metabolite concentration. CONCLUSION:The AC-iTIP approach could probe the chemical exchange effect more robustly than the existing R1? asymmetry acquisition approaches. Therefore, AC-iTIP is a promising technique for metabolite imaging based on the chemical exchange effect.
Project description:Studying protein dynamics on microsecond-to-millisecond (?s-ms) time scales can provide important insight into protein function. In magic-angle-spinning (MAS) NMR, ?s dynamics can be visualized by <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msub><mml:mi>R</mml:mi> <mml:mrow><mml:mn>1</mml:mn> <mml:mi>?</mml:mi></mml:mrow> </mml:msub> </mml:math> rotating-frame relaxation dispersion experiments in different regimes of radio-frequency field strengths: at low RF field strength, isotropic-chemical-shift fluctuation leads to "Bloch-McConnell-type" relaxation dispersion, while when the RF field approaches rotary resonance conditions bond angle fluctuations manifest as increased <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msub><mml:mi>R</mml:mi> <mml:mrow><mml:mn>1</mml:mn> <mml:mi>?</mml:mi></mml:mrow> </mml:msub> </mml:math> rate constants ("Near-Rotary-Resonance Relaxation Dispersion", NERRD). Here we explore the joint analysis of both regimes to gain comprehensive insight into motion in terms of geometric amplitudes, chemical-shift changes, populations and exchange kinetics. We use a numerical simulation procedure to illustrate these effects and the potential of extracting exchange parameters, and apply the methodology to the study of a previously described conformational exchange process in microcrystalline ubiquitin.
Project description:NMR relaxation dispersion methods provide a holistic way to observe microsecond time-scale protein backbone motion both in solution and in the solid state. Different nuclei (1H and 15N) and different relaxation dispersion techniques (Bloch-McConnell and near-rotary-resonance) give complementary information about the amplitudes and time scales of the conformational dynamics and provide comprehensive insights into the mechanistic details of the structural rearrangements. In this paper, we exemplify the benefits of the combination of various solution- and solid-state relaxation dispersion methods on a microcrystalline protein (?-spectrin SH3 domain), for which we are able to identify and model the functionally relevant conformational rearrangements around the ligand recognition loop occurring on multiple microsecond time scales. The observed loop motions suggest that the SH3 domain exists in a binding-competent conformation in dynamic equilibrium with a sterically impaired ground-state conformation both in solution and in crystalline form. This inherent plasticity between the interconverting macrostates is compatible with a conformational-preselection model and provides new insights into the recognition mechanisms of SH3 domains.
Project description:NMR relaxation dispersion studies have shown that Watson-Crick G-C and A-T base pairs in duplex DNA exist in dynamic equilibrium with their Hoogsteen counterparts. Hoogsteen base pairs form through concurrent rotation of the purine base about the glycosidic bond from an anti to a syn conformation and constriction of the C1'-C1' distance across the base pair by ?2?Å to allow Hoogsteen type hydrogen bonding. Owing to their unique structure, Hoogsteen base pairs can play important roles in DNA recognition, the accommodation, recognition, and repair of DNA damage, and in DNA replication. NMR relaxation dispersion experiments targeting imino nitrogen and protonated base and sugar carbons have provided insights into many structural features of transient Hoogsteen base pairs, including one of two predicted hydrogen bonds involving (G)N7···H-N3(C)+ and (A)N7···H-N3(T). Here, through measurement of cytosine amino (N4) R1? relaxation dispersion, we provide direct evidence for the second (G)O6···H2-N4(C)+ hydrogen bond in G(syn)-C+ transient Hoogsteen base pairs. The utility of cytosine N4 R1? relaxation dispersion as a new sensitive probe of transient Hoogsteen base pairs, and cytosine dynamics in general, is further demonstrated by measuring G(syn)-C+ Hoogsteen exchange near neutral pH and in the context of the naturally occurring DNA modification 5-methyl cytosine (m5C), in DNA samples prepared using chemical synthesis and a 15N labeled m5C phosphoramidite.
Project description:High field MRI is beneficial for chemical exchange saturation transfer (CEST) in terms of high SNR, CNR, and chemical shift dispersion. These advantages may, however, be counter-balanced by the increased transmit field inhomogeneity normally associated with high field MRI. The relatively high sensitivity of the CEST contrast to B1 inhomogeneity necessitates the development of correction methods, which is essential for the clinical translation of CEST. In this work, two B1 correction algorithms for the most studied CEST effects, amide-CEST and nuclear Overhauser enhancement (NOE), were analyzed. Both methods rely on fitting the multi-pool Bloch-McConnell equations to the densely sampled CEST spectra. In the first method, the correction is achieved by using a linear B1 correction of the calculated amide and NOE CEST effects. The second method uses the Bloch-McConnell fit parameters and the desired B1 amplitude to recalculate the CEST spectra, followed by the calculation of B1 -corrected amide and NOE CEST effects. Both algorithms were systematically studied in Bloch-McConnell equations and in human data, and compared with the earlier proposed ideal interpolation-based B1 correction method. In the low B1 regime of 0.15-0.50 ?T (average power), a simple linear model was sufficient to mitigate B1 inhomogeneity effects on a par with the interpolation B1 correction, as demonstrated by a reduced correlation of the CEST contrast with B1 in both the simulations and the experiments.
Project description:We investigated correlated µs-ms time scale motions of neighboring 13C'-15N and 13C?-13C? nuclei in both protonated and perdeuterated samples of GB3. The techniques employed, NMR relaxation due to cross-correlated chemical shift modulations, specifically target concerted changes in the isotropic chemical shifts of the two nuclei associated with spatial fluctuations. Field-dependence of the relaxation rates permits identification of the parameters defining the chemical exchange rate constant under the assumption of a two-site exchange. The time scale of motions falls into the intermediate to fast regime (with respect to the chemical shift time scale, 100-400 s-1 range) for the 13C'-15N pairs and into the slow to intermediate regime for the 13C?-13C? pairs (about 150 s-1). Comparison of the results obtained for protonated and deuterated GB3 suggests that deuteration has a tendency to reduce these slow scale correlated motions, especially for the 13C?-13C? pairs.
Project description:Proteins perform their functions in solution but their structures are most frequently studied inside crystals. Here we probe how the crystal packing alters microsecond dynamics, using solid-state NMR measurements and multi-microsecond MD simulations of different crystal forms of ubiquitin. In particular, near-rotary-resonance relaxation dispersion (NERRD) experiments probe angular backbone motion, while Bloch-McConnell relaxation dispersion data report on fluctuations of the local electronic environment. These experiments and simulations reveal that the packing of the protein can significantly alter the thermodynamics and kinetics of local conformational exchange. Moreover, we report small-amplitude reorientational motion of protein molecules in the crystal lattice with an ~3-5° amplitude on a tens-of-microseconds time scale in one of the crystals, but not in others. An intriguing possibility arises that overall motion is to some extent coupled to local dynamics. Our study highlights the importance of considering the packing when analyzing dynamics of crystalline proteins.X-ray crystallography is the main method for protein structure determination. Here the authors combine solid-state NMR measurements and molecular dynamics simulations and show that crystal packing alters the thermodynamics and kinetics of local conformational exchange as well as overall rocking motion of protein molecules in the crystal lattice.
Project description: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 (R1?) 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 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.