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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...

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