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Global and local mobility of apocalmodulin monitored through fast-field cycling relaxometry.

ABSTRACT: Calmodulin (CaM) is a ubiquitous eukaryotic protein with two conformationally independent domains that can bind up to two calcium ions each. In the calcium-bound state, CaM is able to regulate a vast number of cellular activities by binding to a multiplicity of target proteins in different modes. Its versatility has been ascribed to its anomalously high flexibility. The calcium-free form (apoCaM), which is the resting state of CaM in cells, is also able to functionally bind a number of protein targets, but its dynamics has received less attention. At variance with the calcium-bound form, the crystal structure of apoCaM shows a compact organization of the two domains, but NMR measurements could not detect any contact between them, thus indicating the presence of mobility in solution. The mobility of apoCaM is here investigated through protein proton relaxation rate measurements performed with a high-sensitivity fast-field cycling relaxometer. Such measurements provide direct access to the spectral density function and show that 1), the reorientation time is in agreement with a closed form of the protein; but 2), the collective order parameter is much smaller than for other well folded compact proteins, indicating that a remarkably large side-chain mobility must be present.

SUBMITTER: Borsi V

PROVIDER: S-EPMC2749786 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Global and local mobility of apocalmodulin monitored through fast-field cycling relaxometry.

Borsi Valentina V Luchinat Claudio C Parigi Giacomo G

Biophysical journal 20090901 6

Calmodulin (CaM) is a ubiquitous eukaryotic protein with two conformationally independent domains that can bind up to two calcium ions each. In the calcium-bound state, CaM is able to regulate a vast number of cellular activities by binding to a multiplicity of target proteins in different modes. Its versatility has been ascribed to its anomalously high flexibility. The calcium-free form (apoCaM), which is the resting state of CaM in cells, is also able to functionally bind a number of protein t ...[more]

PMID: 19751682

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Project description:The remarkable power and specificity of enzyme catalysis rely on the dynamic alignment of the enzyme, substrates, and cofactors, yet the role of dynamics has usually been approached from the perspective of the protein. We have been using an underappreciated NMR technique, subtesla high-resolution field cycling 31P NMR relaxometry, to investigate the dynamics of the enzyme-bound substrates and cofactor on guanosine-5'-monophosphate reductase (GMPR). GMPR forms two dead end, yet catalytically competent, complexes that mimic distinct steps in the catalytic cycle: E·IMP·NADP+ undergoes a partial hydride transfer reaction, while E·GMP·NADP+ undergoes a partial deamination reaction. A different cofactor conformation is required for each partial reaction. Here we report the effects of mutations designed to perturb cofactor conformation and ammonia binding with the goal of identifying the structural features that contribute to the distinct dynamic signatures of the hydride transfer and deamination complexes. These experiments suggest that Asp129 is a central cog in a dynamic network required for both hydride transfer and deamination. In contrast, Lys77 modulates the conformation and mobility of substrates and cofactors in a reaction-specific manner. Thr105 and Tyr318 are part of a deamination-specific dynamic network that includes the 2'-OH of GMP. These residues have comparatively little effect on the dynamic properties of the hydride transfer complex. These results further illustrate the potential of high-resolution field cycling NMR relaxometry for the investigation of ligand dynamics. In addition, exchange experiments indicate that NH3/NH4+ has a high affinity for the deamination complex but a low affinity for the hydride transfer complex, suggesting that the movement of ammonia may gate the cofactor conformational change. Collectively, these experiments reinforce the view that the enzyme, substrates, and cofactor are linked in intricate, reaction-specific, dynamic networks and demonstrate that distal portions of the substrates and cofactors are critical features in these networks.

Dataset Information

Global and local mobility of apocalmodulin monitored through fast-field cycling relaxometry.

Publications

Global and local mobility of apocalmodulin monitored through fast-field cycling relaxometry.

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