The 2D NMR Experiments H(C)CO2and H[formula omitted]2for Assignment and pH Titration of Carboxylate Groups in Uniformly15N/13C-Labeled Proteins

Abstract

In the practice of assigning the H NMR spectra of prosimultaneously as single-quantum coherence and are observed in a common dimension; CO2 is used to express that teins as a basis for three-dimensional structure determination (1) , unique and complete identification of the amino-acid carboxylates are selectively observed) is derived from the 3D ct-HCACO experiment (13, 14) designed for backboneside-chain spin systems remains a limiting factor. When using homonuclear H NMR, only 6 out of the 20 proteinogenic resonance assignments. The 2D H(C)CO2 experiment is obtained with the same pulse scheme when the C /g chemiamino acids can be uniquely assigned from the symmetry of the spin systems of the nonlabile hydrogen atoms (2) , cal-shift evolution is omitted by setting the factor k to zero (Fig. 1) . In the following product-operator description (15) , and early attempts at editing the H NMR spectra for unique H spin system types (3) found little practical use. In unionly terms resulting in observable magnetization during the detection period are retained and constant multiplicative facformly C/N-labeled proteins studied in solvents where the labile side-chain amide protons are observable, unique tors and trigonometric terms are omitted. Pulsed field gradients (PFG) are employed for coherence pathway rejection scalar coupling patterns can be outlined for all common amino acids, with the possible exception of the residue pair and water suppression (16, 17) . For simplicity, the transfer amplitude will be derived for an Asp residue with degenerate Ser and Cys. To expand the existing arsenal of experiments capable of identifying the complete spin systems in the H chemical shifts. The spin operators for H , C , and CO2 are denoted as I , C , and C*. The experiment starts highly complex protein NMR spectra, this Communication presents two-dimensional triple-resonance NMR experiwith an INEPT transfer of polarization from protons to carbons, so that at time a (Fig. 1) we have transverse C ments for identification of the acidic side-chain spin systems magnetization in antiphase with respect to one of the by selective correlation of CH 2 and CO 2 of Asp, and attached protons, CH 2 and CO d 2 of Glu, and of the C-terminal residue by selective correlation of CH with CO2. Since these experiments, named 2D H(C)CO2 and 2D HCCO2, specifically s(a) A CyIz . [1] select for CO2, they yield greatly simplified spectra. In addition to the use for resonance assignments, an attractive This magnetization is refocused during t2 A 4{JCH} (18) . application is for the determination of the carboxylate pKa Then, the C magnetization is transferred to the adjacent values in investigations of structural roles of the acidic side CO2 group during t3 . To minimize losses due to dephasing chains (4, 5) and their participation in functionally active caused by the passive J(C , C) coupling, t3 is set to sites. 7.2 ms (13, 14) . The magnetization before the first 907 pulse The experiments described presently are an addition to a on CO2 is thus described by CyC *z . Between the time points group of measurements previously introduced for identificaa and b , the CO2 and C chemical shifts evolve during tion of different amino acid side chains, which filter the the times t1 and k 1 t1 ( in a constant time fashion), respecresonances of selected amino acids on the basis of unique tively, so that the magnetization at point b (Fig. 1) is spin–spin scalar couplings (6–10) . Quite naturally, as pointed out by Gehring and Guittet (9) , the resulting spectra s(b) are sparsely populated with peaks and therefore particularly A CyC *z {cos[V(C)k 1 t1]cos[V(CO) t1]}. [2] suited for a reduction in dimensionality by use of the recently published projection technique (11, 12) . The pulse scheme of the 2D HCCO2 experiment (Fig. 1; the underlined letters During the CO2 chemical-shift evolution, C is decoupled by a 1807 pulse. In contrast to the Asp resonances, the scalar indicate that CO2 and its directly attached C spins evolve