Carbonyl ¹³C transverse relaxation measurements to sample protein backbone dynamics

TY - JOUR

T1 - Carbonyl 13C transverse relaxation measurements to sample protein backbone dynamics

AU - Mulder, Frans A A

AU - Akke, Mikael

PY - 2003/10/1

Y1 - 2003/10/1

N2 - Carbonyl 13C relaxation experiments to study protein backbone dynamics have recently been developed. However, the effect of three-bond 13C′-13C′ couplings on transverse relaxation measurements appears not to have been considered, and the potential to detect and quantify motions on the millisecond to microsecond time scale has not been fully explored. The present paper addresses these two issues. Simulations and experiments show that scalar couplings between adjacent backbone carbonyl carbon nuclei and between backbone and side-chain carbonyl/carboxyl carbon atoms in Asp and Asn residues interfere with the accurate determination of transverse relaxation rates by Carr-Purcell-Meiboom-Gill or on-resonance spin-lock measurements. The use of off-resonance radio-frequency fields avoids efficient cross-polarization, and offers a route towards accurate R 1ρ measurements. In addition, this approach yields dispersion in the transverse relaxation rate as a function of the effective field when conformational exchange is present. In the case of calcium-bound calbindin D9k, 13C′ off-resonance R1ρp measurements yielded uniform values of R2 along the polypeptide chain, indicating homogeneous chemical shift anisotropies and restricted dynamics on the picosecond to nanosecond time scale. Variation of R2 as a function of the effective spin-lock field strength was not observed for any residue, indicating the absence of large-scale conformational changes of the protein backbone in the millisecond to microsecond time window. The absence of relaxation induced by internal motions on these wide-ranging time scales reinforces the view that calcium-loaded calbindin D9k is extremely rigid. In contrast, for the C-terminal tryptic fragment of calmodulin containing the E140Q mutation we observed widespread exchange broadening. From the carbonyl transverse relaxation dispersion profile of Asp129 the exchange rate was determined to be 28 000 s-1.

AB - Carbonyl 13C relaxation experiments to study protein backbone dynamics have recently been developed. However, the effect of three-bond 13C′-13C′ couplings on transverse relaxation measurements appears not to have been considered, and the potential to detect and quantify motions on the millisecond to microsecond time scale has not been fully explored. The present paper addresses these two issues. Simulations and experiments show that scalar couplings between adjacent backbone carbonyl carbon nuclei and between backbone and side-chain carbonyl/carboxyl carbon atoms in Asp and Asn residues interfere with the accurate determination of transverse relaxation rates by Carr-Purcell-Meiboom-Gill or on-resonance spin-lock measurements. The use of off-resonance radio-frequency fields avoids efficient cross-polarization, and offers a route towards accurate R 1ρ measurements. In addition, this approach yields dispersion in the transverse relaxation rate as a function of the effective field when conformational exchange is present. In the case of calcium-bound calbindin D9k, 13C′ off-resonance R1ρp measurements yielded uniform values of R2 along the polypeptide chain, indicating homogeneous chemical shift anisotropies and restricted dynamics on the picosecond to nanosecond time scale. Variation of R2 as a function of the effective spin-lock field strength was not observed for any residue, indicating the absence of large-scale conformational changes of the protein backbone in the millisecond to microsecond time window. The absence of relaxation induced by internal motions on these wide-ranging time scales reinforces the view that calcium-loaded calbindin D9k is extremely rigid. In contrast, for the C-terminal tryptic fragment of calmodulin containing the E140Q mutation we observed widespread exchange broadening. From the carbonyl transverse relaxation dispersion profile of Asp129 the exchange rate was determined to be 28 000 s-1.

KW - C NMR

KW - Backbone

KW - Calbindin

KW - Calmodulin

KW - Carbonyl

KW - CPMG

KW - Dynamics

KW - Exchange

KW - NMR

U2 - 10.1002/mrc.1252

DO - 10.1002/mrc.1252

M3 - Journal article

AN - SCOPUS:0242299095

SN - 0749-1581

VL - 41

SP - 853

EP - 865

JO - Magnetic Resonance in Chemistry

JF - Magnetic Resonance in Chemistry

IS - 10

ER -