Single Molecule Conformational Dynamics of Adenylate Kinase: Energy Landscape, Structural Correlations, and Transition State Ensembles

We developed a coarse grained two-well model to study the single molecule protein conformational dynamics in microscopic detail at the residue level, overcoming the often encountered computational bottleneck. In particular, we explored the underlying conformational energy landscape of adenylate kinase, a crucial protein for signal transduction in the cell, and identified two major kinetic pathways for the conformational switch between open and closed states through either the intermediate state or the transient state. Based on the parameters fitted to the room-temperature experimental data, we predicted open and closed kinetic rates at the whole temperature ranges from 10 to 50 °C, which agree well with the experimental turnover numbers. After uncovering the underlying mechanism for conformational dynamics and exploring the structural correlations, we found the crucial dynamical interplay between the nucleoside monophosphate binding domain (NMP) and the ATP-binding domain (LID) in controlling the conformational switch. The key residues and contacts responsible for the conformational transitions are identified by following the time evolution of the two-dimensional spatial contact maps and characterizing the transition state as well as intermediate structure ensembles through φ value analysis. Our model provides a general framework to study the conformational dynamics of biomolecules and can be applied to many other systems

Single-Molecule Fluorescence and Spectroscopy Studies of Cdc42-CBD Protein–Protein Interaction Dynamics

<div><p>(A) Second-order auto-correlation function, C(t), calculated from a fluorescence intensity trajectory I(t) of a single Cdc42-CBD complex. Solid curve is a bi-exponential fit. (Inset) A single-molecule fluorescence raster-scanning image of GTP-loaded Cdc42 in complex with dye-labeled CBD biosensor.</p><p>(B) The occurrence histogram of the single-complex conformational fluctuation rate for 60 individual protein complexes [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020078#pcbi-0020078-b019" target="_blank">19</a>].</p></div

Two-Dimensional Free-Energy Landscape versus Interface Binding of Cdc42 and Folding CBD for Different α

<p>Two-Dimensional Free-Energy Landscape versus Interface Binding of Cdc42 and Folding CBD for Different α</p

Illustration of Flexible Binding-Folding through Two-Dimensional Free-Energy Landscape, Trajectory and Time Correlation

<div><p>(A) Flexible binding-folding.</p><p>(B) Two-dimensional free-energy contour plots of the interface degree of freedom between CBD portion of WASP and Cdc42, and conformation/folding degree of freedom for CBD.</p><p>(C) Typical trajectory of fraction of native contacts of binding interface between CBD portion of WASP and Cdc42.</p><p>(D) Time correlation function of fraction of native interface contacts.</p></div

Distribution of Phi Values and Phi Values versus Protein-Primary Sequence for Different α

<p>Distribution of Phi Values and Phi Values versus Protein-Primary Sequence for Different α</p

Heat Capacity <i>C_v</i>(<i>T</i>)

<p>The simulation temperature <i>T_s</i> = 0.944, and melting temperature <i>T_f</i> = 1.0. The <i>T_f</i> is defined from the peak of <i>C_v</i>, and <i>T_s</i> is determined from two-dimensional free-energy profile <i>F</i>(<i>Q_f</i>₂,<i>Q_b</i>)<b>.</b> The simulation temperature was chosen so that the free-energy values at the loosely bound and bound states are equal.</p

Distribution of Phi Values versus Protein-Primary Sequence

<p>(A) Distribution of phi values, and (B) phi values along the primary sequences of CBD portion of WASP and Cdc42.</p

Auto-Correlations of Fraction of Interface Contacts <i>c</i>(Δ) at Different Positions of <i>Q_b</i> and at <i>T</i> = <i>T_s</i>

<p>Auto-Correlations of Fraction of Interface Contacts <i>c</i>(Δ) at Different Positions of <i>Q_b</i> and at <i>T</i> = <i>T_s</i></p

Typical Structures and their Corresponding Configuration in the Free-Energy Landscape

<p>Typical structures of loosely bound state (left structure), transition state (middle structure), and bound state (right structure) and their corresponding configuration in free-energy profiles in terms of fraction of native (interface) contacts between Cdc42 and CBD portion of WASP, as well as the fraction of native (folding) contacts of CBD itself.</p

An Illustration of Two State Cdc42-CBD Binding

<p>The loosely bound state was presumably a subset of conformations that deviated from the bound equilibrium states, probed by the dye, without disrupting the subnanometer long-range interactions, so the overall protein complex was still associated.</p