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Mechanistic details of a protein-protein association pathway revealed by paramagnetic relaxation enhancement titration measurements.

ABSTRACT: Protein-protein association generally proceeds via the intermediary of a transient, lowly populated, encounter complex ensemble. The mechanism whereby the interacting molecules in this ensemble locate their final stereospecific structure is poorly understood. Further, a fundamental question is whether the encounter complex ensemble is an effectively homogeneous population of nonspecific complexes or whether it comprises a set of distinct structural and thermodynamic states. Here we use intermolecular paramagnetic relaxation enhancement (PRE), a technique that is exquisitely sensitive to lowly populated states in the fast exchange regime, to characterize the mechanistic details of the transient encounter complex interactions between the N-terminal domain of Enzyme I (EIN) and the histidine-containing phosphocarrier protein (HPr), two major bacterial signaling proteins. Experiments were conducted at an ionic strength of 150 mM NaCl to eliminate any spurious nonspecific associations not relevant under physiological conditions. By monitoring the dependence of the intermolecular transverse PRE (Gamma(2)) rates measured on (15)N-labeled EIN on the concentration of paramagnetically labeled HPr, two distinct types of encounter complex configurations along the association pathway are identified and dissected. The first class, which is in equilibrium with and sterically occluded by the specific complex, probably involves rigid body rotations and small translations near or at the active site. In contrast, the second class of encounter complex configurations can coexist with the specific complex to form a ternary complex ensemble, which may help EIN compete with other HPr binding partners in vivo by increasing the effective local concentration of HPr even when the active site of EIN is occupied.

SUBMITTER: Fawzi NL

PROVIDER: S-EPMC2824347 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Mechanistic details of a protein-protein association pathway revealed by paramagnetic relaxation enhancement titration measurements.

Fawzi Nicolas L NL Doucleff Michaeleen M Suh Jeong-Yong JY Clore G Marius GM

Proceedings of the National Academy of Sciences of the United States of America 20100107 4

Protein-protein association generally proceeds via the intermediary of a transient, lowly populated, encounter complex ensemble. The mechanism whereby the interacting molecules in this ensemble locate their final stereospecific structure is poorly understood. Further, a fundamental question is whether the encounter complex ensemble is an effectively homogeneous population of nonspecific complexes or whether it comprises a set of distinct structural and thermodynamic states. Here we use intermole ...[more]

PMID: 20080627

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Project description:Longitudinal (Γ1) and transverse (Γ2) solvent paramagnetic relaxation enhancement (sPRE) yields field-dependent information in the form of spectral densities that provides unique information related to cosolute-protein interactions and electrostatics. A typical protein sPRE data set can only sample a few points on the spectral density curve, J(ω), within a narrow frequency window (500 MHz to ∼1 GHz). However, complex interactions and dynamics of paramagnetic cosolutes around a protein make it difficult to directly interpret the few experimentally accessible points of J(ω). In this paper, we show that it is possible to significantly extend the experimentally accessible frequency range (corresponding to a range from ∼270 MHz to 1.8 GHz) by acquiring a series of sPRE experiments at different temperatures. This approach is based on the scaling property of J(ω) originally proposed by Melchior and Fries for small molecules. Here, we demonstrate that the same scaling property also holds for geometrically far more complex systems such as proteins. Using the extended spectral densities derived from the scaling property as the reference dataset, we demonstrate that our previous approach that makes use of a non-Lorentzian Ansatz spectral density function to fit only J(0) and one to two J(ω) points allows one to obtain accurate values for the concentration-normalized equilibrium average of the electron-proton interspin separation ⟨r-6⟩norm and the correlation time τC, which provide quantitative information on the energetics and timescale, respectively, of local cosolute-protein interactions. We also show that effective near-surface potentials, ϕENS, obtained from ⟨r-6⟩norm provide a reliable and quantitative measure of intermolecular interactions including electrostatics, while ϕENS values obtained from only Γ1 or Γ2 sPRE rates can have significant artifacts as a consequence of potential variations and changes in the diffusive properties of the cosolute around the protein surface. Finally, we discuss the experimental feasibility and limitations of extracting the high-frequency limit of J(ω) that is related to ⟨r-8⟩norm and report on the extremely local intermolecular potential.

Dataset Information

Mechanistic details of a protein-protein association pathway revealed by paramagnetic relaxation enhancement titration measurements.

Publications

Mechanistic details of a protein-protein association pathway revealed by paramagnetic relaxation enhancement titration measurements.

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