A Relaxation-Compensated Carr−Purcell−Meiboom−Gill Sequence for Characterizing Chemical Exchange by NMR Spectroscopy

Abstract

Functions of biological macromolecules, including enzyme catalysis, allostery, and ligand recognition, in some circumstances can be rate-limited by dynamic processes that couple nonproductive and productive conformations. 1,2 Delineating the amplitudes, rates, and mechanisms of these motions is necessary to explicate biological function. 3-5 This paper describes a modified protondetected 15 N Carr-Purcell-Meiboom-Gill 6,7 (CPMG) spin-echo pulse sequence that overcomes the disadvantages associated with pulsing rates comparable to the one-bond J NH scalar coupling constant. The new experiment permits measurement of chemical or conformational exchange time constants from approximately 0.5 to 5 ms. This range of time scales fills an important gap between microsecond time scales that are accessible by the offresonance rotating-frame experiment 8,9 and millisecond time scales that are accessible by the zz-exchange experiment. [10][11][12] Conformational or chemical kinetic processes that transfer a nuclear spin between sites with different magnetic environments contribute to the adiabatic decay of transverse magnetization. The phenomenological transverse relaxation rate constant measured in a CPMG experiment, R 2 (τ cp ), is given by:in which τ cp is the delay between pulses in the spin-echo pulse train; R in and R anti are the transverse relaxation rate constants for in-phase and antiphase coherences averaged over the populations of each chemical or conformational state, respectively; 0 e e 1 reflects the averaging between in-phase and antiphase coherences due to evolution under the scalar coupling Hamiltonian during τ cp ; 13-15 and R ex is the rate constant for damping due to exchange between sites. For two exchanging sites 16 in which Φ ex ) (ω 1 -ω 2 ) 2 p 1 p 2 ; and p i and ω i are the populations and Larmor frequencies for the nuclear spin in site i; and τ ex is the reduced lifetime of the exchanging sites.In principle, exchange kinetics can be determined by fitting eqs 1 and 2 to R 2 (τ cp ) data obtained for different values of τ cp ; however, both and R ex depend on τ cp , which renders the analysis intractable. This difficulty is circumvented if τ cp is limited to small values (<1/(4J NH )), because ≈ 1. 17 In this case, only fast exchange processes can be studied, and the resulting high radio frequency duty cycle poses a significant drawback compared with the off-resonance rotating-frame experiment. 8,9 Broadband proton decoupling during τ cp prevents evolution of in-phase into antiphase magnetization and makes ) 1; however, decoupling interferes with spin-echo formation and accentuates the decay of transverse magnetization. 14,15,18 In the approach adopted herein, the rate constants for in-phase and antiphase coherences are averaged explicitly during the CPMG sequence to render ) 0.5 for all τ cp . Application of the experiment to basic pancreatic trypsin inhibitor (BPTI) confirms the effectiveness of the proposed averaging procedure for values of τ cp as large as 2/J NH .The modified CPMG pulse sequenc...

NMR studies of dynamics in RNA and DNA by 13C relaxation

“…Thus, motions in the ns-s range are undetectable by relaxation measurements, although slower motions at rates comparable to the chemical shift differences between conformational states that are sampled by these motional processes are observed as modulations of the linewidths. Slower ( s-ms) motions can thus be characterized either by relaxation dispersion [30][31][32] or the power dependence of T 1 . 33,34 …”

PhosphoThr Peptide Binding Globally Rigidifies Much of the FHA Domain from Arabidopsis Receptor Kinase-Associated Protein Phosphatase,

“…For good water suppression with cryogenic probes and economy of pulses for the 8 mm probe, home-implemented 3-9-19 WATERGATE (49) versions of sequences of ref (50) were used for 15 N{ 1 H} steady-state NOE, 500 MHz R 1 and R 2 . The relaxation-compensated CPMG pulse sequence of ref (51) was applied to the free state of KI-FHA and the TROSY-enhanced version (52) to the bound state, using the Inova 600 for each. τ cp delays were in both cases set for 2 ms and 7.5 ms π pulse spacing, to identify sites of exchange broadening on the msec scale.…”

“…Using relaxation-compensated CPMG sequences with τ cp = 1 ms (π pulse spacing of 2 ms), total relaxation delays were 16, 32, 48, 64, 80, 96 ms using the sequence of ref (51) or 8, 32, 64, 96, 128, and 160 ms using the sequence of ref (52). At τ cp = 3.75 ms (π pulse spacing of 7.5 ms) total delays were 30, 60, 90, and 120 ms using the sequence of ref (51) or 30, 60, 90, 120, 150, and 180 ms using the sequence of ref (52).…”

“…At τ cp = 3.75 ms (π pulse spacing of 7.5 ms) total delays were 30, 60, 90, and 120 ms using the sequence of ref (51) or 30, 60, 90, 120, 150, and 180 ms using the sequence of ref (52). Relaxation delay periods at 500 MHz were 20 * , 60, 120 * , 240, 400 * , 600 and 860 * ms for R 1 and 16 * , 32, 48 * , 64, 80 * , 112, 144 * ms for R 2 .…”

“…(The term β-hairpin refers to both anti-parallel strands and the intervening loop). Relaxation-compensated CPMG R 2 measurements comparing averaged R 2 between faster and slower pulsing of CPMG π pulses can exaggerate the line broadening of msec-scale chemical exchange processes (51). Elevated ΔR 2 = R 2 (7.5 ms) − R 2 (2 ms) in Figure 1d clearly suggest msec exchange broadening to occur in the 1/2 β-hairpin, the 4/5 loop, β8, the 9/10 loop, 10/11 β-hairpin, and C-terminus.…”