Increasing the exchange time-scale that can be probed by CPMG relaxation dispersion NMR.
ABSTRACT Carr-Purcell-Meiboom-Gill relaxation dispersion NMR spectroscopy has emerged as a valuable tool to characterize conformational exchange between major and minor states in a large variety of biomolecules. The window of exchange that is amenable for study, corresponding to rates on the order of 2000 s(-1) or less, is limiting, however. Here we show that a combined analysis of both amide (15)N and (1)H(N) CPMG profiles and major state exchange induced (15)N chemical shift changes leads to significant increases in the exchange time scale for which accurate exchange parameters and chemical shift differences between the interconverting states can be obtained. The utility of the approach is illustrated with examples involving a pair of protein systems that are in the moderately fast exchange regime. In these cases the analysis of dispersion profiles alone is not sufficient to obtain robust measures of exchange parameters and chemical shift differences. Inclusion of major state exchange induced (15)N chemical shift changes measured in ((15)N-(1)H(N)) HMQC and HSQC data sets in addition to the (15)N and (1)H(N) dispersion profiles in the analysis "breaks" the correlation in parameters, allowing accurate values to be obtained. The approach is straightforward to implement and makes use of HMQC/HSQC data sets that are recorded as a matter of routine to obtain chemical shifts of the excited state. It promises to increase the range of exchanging systems involving low populated, transiently formed excited states that can be studied by relaxation dispersion NMR.
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ABSTRACT: The visualization of RNA conformational changes has provided fundamental insights into how regulatory RNAs carry out their biological functions. The RNA structural transitions that have been characterized so far involve long-lived species that can be captured by structure characterization techniques. Here we report the nuclear magnetic resonance visualization of RNA transitions towards 'invisible' excited states (ESs), which exist in too little abundance (2-13%) and for too short a duration (45-250 μs) to allow structural characterization by conventional techniques. Transitions towards ESs result in localized rearrangements in base-pairing that alter building block elements of RNA architecture, including helix-junction-helix motifs and apical loops. The ES can inhibit function by sequestering residues involved in recognition and signalling or promote ATP-independent strand exchange. Thus, RNAs do not adopt a single conformation, but rather exist in rapid equilibrium with alternative ESs, which can be stabilized by cellular cues to affect functional outcomes.Nature 10/2012; · 38.60 Impact Factor