Measurement of 1 J NC' and 2 J H N C' Couplings from Spin-State-Selective Two-Dimensional Correlation Spectrum Perttu Permi,* ,1 Sami Heikkinen,* Ilkka Kilpela ¨inen,* and Arto Annila² *Institute of Biotechnology, P.O. Box 56, University of Helsinki, FIN-00014, Helsinki, Finland; and ² VTT Chemical Technology, P.O. Box 1401, FIN-02044 VTT, Espoo, Finland Received January 19, 1999; revised May 17, 1999 A method for the measurement of 1 J NC and 2 J H N C coupling constants from a simplified two-dimensional [ 15 N, 1 H] correlation spectrum is presented. The multiplet components of the 1 J NC doublet in the indirect dimension and 2 J H N C in the direct dimen- sion are separated into two subspectra by spin-state-selective fil- ters. Thus each subspectrum contains no more peaks than the conventional [ 15 N, 1 H]-HSQC spectrum. Furthermore, the method for the measurement of 1 J NC and 2 J H N C is designed to exploit destructive relaxation interference (TROSY). The results are ver- ified against the measurements of 1 J NC from spin-state-selective [ 13 C, 1 H] correlation spectra recorded with additional sequence described here. © 1999 Academic Press Key Words: coupling; spin-state-selective filters; TROSY; ubiquitin. INTRODUCTION Measurement of coupling constants for protein structure determination has remained of interest for several years. E.COSY (1–5), quantitative J -correlation (6 –10), and tradi- tional J -coupled (11–13) experiments have been employed to measure primarily 3 J scalar coupling constants related to di- hedrals via Karplus relations. The traditional J -coupled meth- ods provide an easy and accurate measurement of couplings but suffer from spectral crowding owing to the simultaneous presence of many multiplet components. To alleviate this over- lap problem, editing with respect to a third dimension has been used (4). Also, recently spin-state-selective /-filters have been devised to separate doublet components into two different subspectra (14 –24). These methods have been implemented also in measurements of large heteronuclear one-bond cou- plings from proteins dissolved in weakly oriented liquid crystal phases to determine 1 J H N N , 1 J NC' , and 2 J H N C' with dipolar con- tributions using a 15 N spin-state edited spectrum (25). The ease of extracting orientational information from one- and two-bond couplings, preferably from simplified two-dimensional spectra, is of an obvious importance. In this paper, we show a straightforward but practical ex- tension of the use of spin-state-selective filters to measure 1 J NC' and 2 J H N C' couplings from [ 15 N, 1 H] correlation spectra in 15 N/ 13 C-labeled proteins. We refer to this experiment as HN(/ -NC'-J ). In addition, we provide the selection for the [ 15 N, 1 H] multiplet component with the most favorable relaxation properties (TROSY) (18 –22). This method, referred as HN(/ -NC'-J )-TROSY, is particularly amenable to perdeuterated proteins studied at high magnetic fields. We also introduce another method which allows the determination of 1 J NC' from a two-dimensional spin-state-selective [ 13 C', 1 H] correlation spectrum as an attractive alternative for measurements at in- termediate field strengths. We denote this experiment with the abbreviation H(/-NC'-J )CO. The original /-filters (14– 24) are modified for the present purpose to select carbonyl carbon or nitrogen spin-states. In the HN(/-NC'-J ) correla- tion experiment in- and antiphase 15 N– 13 C' magnetization are selected prior to the t 1 evolution period by the /-filters with respect to 1 J NC' , whereas in the H(/-NC'-J )CO correlation experiment the in- and antiphase magnetization are edited with respect to 1 J H N N . Similar to the S 3 E -filter element, the unde- sired components of transverse magnetization are purged (14, 15). This allows the determination of two different cou- plings from a simplified spectrum with pure absorptive line- shapes. DESCRIPTION OF THE PULSE SEQUENCES The HN(/-NC'-J ) pulse sequence to measure 1 J NC' and 2 J H N C' (Fig. 1A) from a [ 15 N, 1 H] correlation spectrum is based on the conventional [ 15 N, 1 H]-HSQC experiment. Proton mag- netization is first transferred into an antiphase heteronuclear single-quantum coherence between the amide proton and the nitrogen with the usual INEPT-step tuned to 1/(2J H N N ), fol- lowed by the /-filter before the t 1 evolution period. In the first experiment, referred to as the antiphase experiment (filled 180° pulse on 13 C' and 90° pulse on 15 N are applied), the heteronuclear coupling between the amide nitrogen and the adjacent carbonyl carbon evolves during the filter of length 1/(2J NC' ) into an antiphase magnetization with respect to 1 J NC' . In the second experiment, referred to as the in-phase experi- ment (unfilled 180° pulses are applied on 13 C'), 15 N is effec- tively decoupled from 13 C'. During the t 1 evolution period, the 1 To whom correspondence should be addressed: Fax: +358-9-708 59541, E-mail: Perttu.Permi@helsinki.fi. Journal of Magnetic Resonance 140, 32– 40 (1999) Article ID jmre.1999.1817, available online at http://www.idealibrary.com on 32 1090-7807/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.