Communication Accurate determination of small one-bond heteronuclear residual dipolar couplings by F1 coupled HSQC modified with a G-BIRD ðrÞ module Krisztina Feh er, a Stefan Berger, a, * and Katalin E. K€ ov er b a Institute for Analytical Chemistry, University of Leipzig, Johnnisallee 29, D-04103, Leipzig, Germany b Department of Inorganic and Analytical Chemistry, University of Debrecen, Debrecen, Hungary Received 18 December 2002; revised 7 March 2003 Abstract WereportaG-BIRD ðrÞ modifiedcoupledHSQCexperimentfortheaccuratedeterminationofone-bondheteronuclearresidual dipolar couplings. The G-BIRD ðrÞ module has been employed to refocus the long-range coupling evolution of the heteronucleus duringthe t 1 frequencylabelingperiod.Asaresult,thecrosspeaksobtainedaresplitbyonlythedirectone-bondcouplingthatcan beextractedbymeasuringsimplefrequencydifferencesbetweensingletmaxima.Additionallythedecouplingoflong-rangemultiple bond splittings leads to considerable sensitivity enhancement. The modification also has been applied in a TROSY sequence re- sulting in a significant sensitivity and resolution improvement. Ó 2003 Published by Elsevier Science (USA). Keywords: Residual dipolar coupling constant; One-bond heteronuclear coupling constant; Coupled HSQC; G-BIRD; TROSY 1. Introduction Residual dipolar couplings (RDC) provide unique long-range constraints for structure determination of molecules conveying information on the distance be- tween nuclei in dipolar interaction and on the orienta- tions of the corresponding internuclear bond vectors relative to the molecular alignment tensor. In recent years residual dipolar couplings have therefore been extensively applied for structural studies of proteins, nucleic acids and carbohydrates [1] in liquid state and proved to improve the precision of structures. Dipolar couplings are orientation dependent tensorial NMR parameters that are averaged to zero by the random tumbling of molecules in isotropic media. RDCÕs arise from the anisotropic tumbling of molecules resulting from their partial alignment in the applied medium. Intrinsic partial alignment is observed for molecules possessing sufficiently large magnetic susceptibility an- isotropy such as nucleic acids, protein–nucleic acid complexes,andmetalbindingproteinsinthepresenceof strongmagneticfield.Atunabledegreeofalignmentcan also be achieved by dissolving the molecule in partially oriented media such as a dilute liquid crystalline phase. RDCÕs manifest themselves in the NMR spectra as an increase or decrease of the splittings due to scalar cou- plingsbetweennuclei.Theirmagnitudecanthereforebe extractedbymeasuringchangesofsplittingsinisotropic versus anisotropic sample conditions. For small flexible molecules the RDCÕs are often smallduetotheeffectsofdynamics[2].Sphericalshape of the molecules may also result in low degree of alignment yielding only small RDCÕs [3–4]. In these cases special considerations have to be taken in the choice of the measurement technique. Herein we propose a modification of the F1-coupled HSQCexperimentwhereadditionalsplittingscausedby long-range multiple bond couplings are eliminated with the aid of G-BIRD ðrÞ module inserted in the heteronu- cleus frequency labelling period. Thus one-bond het- eronuclear RDCÕscanbedeterminedfromtheresulting spectra by simply measuring the frequency difference between the peak maxima of singlets instead of the Journal of Magnetic Resonance 163 (2003) 340–346 www.elsevier.com/locate/jmr * Corresponding author. Fax: +49-341-9736115. E-mail address: stberger@rz.uni-leipzig.de (S. Berger). 1090-7807/$ - see front matter Ó 2003 Published by Elsevier Science (USA). doi:10.1016/S1090-7807(03)00113-7