J. Patrick Loria

ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTA Relaxation-Compensated Carr−Purcell−Meiboom−Gill Sequence for Characterizing Chemical Exchange by NMR SpectroscopyJ. Patrick Loria, Mark Rance, and Arthur G. PalmerView Author Information Department of Biochemistry and Molecular Biophysics Columbia University, New York, New York 10032 Department of Molecular Genetics Biochemistry, and Microbiology University of Cincinnati, Cincinnati, Ohio 45267 Cite this: J. Am. Chem. Soc. 1999, 121, 10, 2331–2332Publication Date (Web):February 26, 1999Publication History Received17 November 1998Revised3 February 1999Published online26 February 1999Published inissue 1 March 1999https://pubs.acs.org/doi/10.1021/ja983961ahttps://doi.org/10.1021/ja983961arapid-communicationACS PublicationsCopyright © 1999 American Chemical SocietyRequest reuse permissionsArticle Views5348Altmetric-Citations571LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-AlertscloseSupporting Info (1)»Supporting Information Supporting Information SUBJECTS:Colloids,Kinetic parameters,Magnetic properties,Mathematical methods,Quantum mechanics Get e-Alerts

The static magnetic field dependence of chemical exchange linebroadening in NMR spectroscopy is investigated theoretically and experimentally. Two-site exchange (A ⇆ B) is considered with site A more highly populated than site B (pa > pb), a shift difference between sites equal to Δω, and an exchange rate constant given by kex. The exchange contribution to the transverse relaxation rate constant for the more highly populated site is denoted Rex. The dependence of Rex on the static magnetic field strength is characterized by a scaling parameter α = d ln Rex/d ln Δω, in which 0 ≤ α ≤ 2 for pa > 0.7. The value of α depends on the NMR chemical shift time scale for the exchange process: for slow exchange (kex/Δω < 1), 0 ≤ α < 1; for intermediate exchange (kex/Δω = 1), α = 1; and for fast exchange (kex/Δω > 1), 1 < α ≤ 2. Consequently, the static magnetic field dependence of Rex defines the chemical shift time scale for an exchange process even if the populations are so highly skewed (pa ≫ pb) that the minor resonance is not observable in the slow exchange limit. The theoretical results are verified by measuring 15N transverse relaxation rate constants at static magnetic fields of 11.7 and 14.1 T and temperatures of 300 and 313 K for the protein basic pancreatic trypsin inhibitor. At each combination of static magnetic field and temperature, the rate constants were measured using Carr−Purcell−Meiboom−Gill and Hahn echo techniques with spin−echo delays ranging from 1.0 to 64.5 ms. 15N resonances for residues in the region of the Cys14-Cys38 disulfide bond are broadened due to chemical exchange. Values of α obtained from the relaxation rate constants range from 0.26 ± 0.17 for Arg39 at 300 K to 1.96 ± 0.25 for Cys38 at 313 K. For Cys38 and Arg39, the two residues most strongly affected by chemical exchange, values of kex were determined to be 380 ± 70 s-1 and 530 ± 90 s-1 at 300 K and 1300 ± 290 s-1 and 1370 ± 160 s-1 at 313 K by global analysis of the relaxation rate constants. The scaling parameters α indicate that chemical exchange for most residues in basic pancreatic trypsin inhibitor does not satisfy kex/Δω ≫ 1. Consequently, the assumption of fast-limit quadratic scaling of exchange broadening in proteins and other macromolecules may be incorrect, even if a single broadened resonance is observed for a nuclear spin. The theoretical results for the static magnetic field dependence of chemical exchange broadening in NMR spectroscopy are applicable to other nuclei and to other techniques for measuring chemical exchange linebroadening.

Determining the principal energy-transfer pathways responsible for allosteric communication in biomolecules remains challenging, partially due to the intrinsic complexity of the systems and the lack of effective characterization methods. In this work, we introduce the eigenvector centrality metric based on mutual information to elucidate allosteric mechanisms that regulate enzymatic activity. Moreover, we propose a strategy to characterize the range of correlations that underlie the allosteric processes. We use the V-type allosteric enzyme imidazole glycerol phosphate synthase (IGPS) to test the proposed methodology. The eigenvector centrality method identifies key amino acid residues of IGPS with high susceptibility to effector binding. The findings are validated by solution NMR measurements yielding important biological insights, including direct experimental evidence for interdomain motion, the central role played by helix h[Formula: see text], and the short-range nature of correlations responsible for the allosteric mechanism. Beyond insights on IGPS allosteric pathways and the nature of residues that could be targeted by therapeutic drugs or site-directed mutagenesis, the reported findings demonstrate the eigenvector centrality analysis as a general cost-effective methodology to gain fundamental understanding of allosteric mechanisms at the molecular level.

High-resolution proton-detected heteronuclear correlation NMR spectroscopy allows the measurement of 15N spin relaxation rates at multiple sites throughout a biological macromolecule. The rate constants are determined by stochastic internal motions on time scales of picoseconds to nanoseconds, overall molecular rotational diffusion on time scales of nanoseconds, and chemical exchange rates on time scales of microseconds to milliseconds. A new method has been developed for distinguishing the contributions of chemical exchange from the contributions due to anisotropic rotational diffusion by measuring both longitudinal and transverse interference between the 1H−15N dipolar and 15N chemical shift anisotropy interactions. The spectroscopic experiment for measuring the longitudinal cross-correlation rate constant for 1H−15N dipolar/15N chemical shift anisotropy interference is based on the approach for measuring the transverse cross-correlation rate constant (Tjandra, N.; Szabo, A.; Bax, A. J. Am. Chem. Soc. 1996, 118, 6986−6991) but incorporates a novel method for averaging the relaxation rates of longitudinal magnetization and two spin order. Application of this technique to Escherichia coli ribonuclease H affords an improved description of rotational diffusion anisotropy and permits a more accurate assessment of chemical exchange in this molecule. The results definitively demonstrate that amino acid residues K60 and W90 are subject to conformational exchange processes, whereas increased transverse relaxation rates for residues in the helix αD arise from anisotropic rotational diffusion.