Analysis of Chemical Kinetics of Multistep Reactions by Mean Reaction Time.

Although methods to derive the rate equation from a kinetic model have been known for over a century, it remains mathematically challenging to derive the rate equation for complex reactions involving multiple steps, as the derivation requires a solution for simultaneous differential equations. Furthermore, the derived kinetic equations are often difficult to intuitively understand. Here, we report a radically different approach to analyze chemical kinetics using the mean reaction time, the average of the time required for the completion of a chemical reaction.

Chaperone action of a cofactor in protein folding.

Previously, we have reported that ATP accelerates the folding and unfolding of Escherichia coli glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a glycolytic enzyme utilizing NAD as a cofactor. Because ATP and NAD share the ADP moiety, we hypothesized that NAD also accelerates the folding of GAPDH and that the common structural motif between ATP and NAD is responsible for the chaperone activity. After confirming that NAD indeed accelerates the folding of GAPDH, we examined the chaperone activity of the structural fragments of NAD (ADP, AMP, adenosine, and nicotinamide monophosphate).

A Cooperative Folding Unit as the Structural Link for Energetic Coupling within a Protein.

Previously, we demonstrated that binding of a ligand to Escherichia coli cofactor-dependent phosphoglycerate mutase (dPGM), a homodimeric protein, is energetically coupled with dimerization. The equilibrium unfolding of dPGM occurs with a stable, monomeric intermediate. Binding of several nonsubstrate metabolites stabilizes the dimeric native form over the monomeric intermediate, reducing the population of the intermediate.

GalaxyHomomer: a web server for protein homo-oligomer structure prediction from a monomer sequence or structure.

Homo-oligomerization of proteins is abundant in nature, and is often intimately related with the physiological functions of proteins, such as in metabolism, signal transduction or immunity. Information on the homo-oligomer structure is therefore important to obtain a molecular-level understanding of protein functions and their regulation. Currently available web servers predict protein homo-oligomer structures either by template-based modeling using homo-oligomer templates selected from the protein structure database or by ab initio docking of monomer structures resolved by experiment or predicted by computation.

Discovery of Nicotinamide Adenine Dinucleotide Binding Proteins in the Escherichia coli Proteome Using a Combined Energetic- and Structural-Bioinformatics-Based Approach.

Protein-ligand interaction plays a critical role in regulating the biochemical functions of proteins. Discovering protein targets for ligands is vital to new drug development. Here, we present a strategy that combines experimental and computational approaches to identify ligand-binding proteins in a proteomic scale.

Effect of circular permutations on transient partial unfolding in proteins.

Under native conditions, proteins can undergo transient partial unfolding, which may cause proteins to misfold or aggregate. A change in sequence connectivity by circular permutation may affect the energetics of transient partial unfolding in proteins without altering the three-dimensional structures. Using Escherichia coli dihydrofolate reductase (DHFR) as a model system, we investigated how circular permutation affects transient partial unfolding in proteins.

Energetic Coupling between Ligand Binding and Dimerization in Escherichia coli Phosphoglycerate Mutase.

Energetic coupling of two molecular events in a protein molecule is ubiquitous in biochemical reactions mediated by proteins, such as catalysis and signal transduction. Here, we investigate energetic coupling between ligand binding and folding of a dimer using a model system that shows three-state equilibrium unfolding of an exceptional quality. The homodimeric Escherichia coli cofactor-dependent phosphoglycerate mutase (dPGM) was found to be stabilized by ATP in a proteome-wide screen, although dPGM does not require or utilize ATP for enzymatic function.

Salt bridge as a gatekeeper against partial unfolding.

Salt bridges are frequently observed in protein structures. Because the energetic contribution of salt bridges is strongly dependent on the environmental context, salt bridges are believed to contribute to the structural specificity rather than the stability. To test the role of salt bridges in enhancing structural specificity, we investigated the contribution of a salt bridge to the energetics of native-state partial unfolding in a cysteine-free version of Escherichia coli ribonuclease H (RNase H*).

Bacteriorhodopsin folds through a poorly organized transition state.

The folding mechanisms of helical membrane proteins remain largely uncharted. Here we characterize the kinetics of bacteriorhodopsin folding and employ φ-value analysis to explore the folding transition state. First, we developed and confirmed a kinetic model that allowed us to assess the rate of folding from SDS-denatured bacteriorhodopsin (bRU) and provides accurate thermodynamic information even under influence of retinal hydrolysis.

Ligand binding to a high-energy partially unfolded protein.

The conformational energy landscape of a protein determines populations of all possible conformations of the protein and also determines the kinetics of the conversion between the conformations. Interaction with ligands influences the conformational energy landscapes of proteins and shifts populations of proteins in different conformational states. To investigate the effect of ligand binding on partial unfolding of a protein, we use Escherichia coli dihydrofolate reductase (DHFR) and its functional ligand NADP(+) as a model system.

Product inhibition in native-state proteolysis.

The proteolysis kinetics of intact proteins by nonspecific proteases provides valuable information on transient partial unfolding of proteins under native conditions. Native-state proteolysis is an approach to utilize the proteolysis kinetics to assess the energetics of partial unfolding in a quantitative manner. In native-state proteolysis, folded proteins are incubated with nonspecific proteases, and the rate of proteolysis is determined from the disappearance of the intact protein.

Structure of a partially unfolded form of Escherichia coli dihydrofolate reductase provides insight into its folding pathway.

Proteins frequently fold via folding intermediates that correspond to local minima on the conformational energy landscape. Probing the structure of the partially unfolded forms in equilibrium under native conditions can provide insight into the properties of folding intermediates. To elucidate the structures of folding intermediates of Escherichia coli dihydrofolate reductase (DHFR), we investigated transient partial unfolding of DHFR under native conditions.

Simplified proteomics approach to discover protein-ligand interactions.

Identifying targets of biologically active small molecules is an essential but still challenging task in drug research and chemical genetics. Energetics-based target identification is an approach that utilizes the change in the conformational stabilities of proteins upon ligand binding in order to identify target proteins. Different from traditional affinity-based capture approaches, energetics-based methods do not require any labeling or immobilization of the test molecule.

Selective stabilization of a partially unfolded protein by a metabolite.

When proteins fold in vivo, the intermediates that exist transiently on their folding pathways are exposed to the potential interactions with a plethora of metabolites within the cell. However, these potential interactions are commonly ignored. Here, we report a case in which a ubiquitous metabolite interacts selectively with a nonnative conformation of a protein and facilitates protein folding and unfolding process.

Revisiting the folding kinetics of bacteriorhodopsin.

The elucidation of the physical principles that govern the folding and stability of membrane proteins is one of the greatest challenges in protein science. Several insights into the folding of α-helical membrane proteins have come from the investigation of the conformational equilibrium of H. halobium bacteriorhodopsin (bR) in mixed micelles using SDS as a denaturant.

Thermodynamic stability of bacteriorhodopsin mutants measured relative to the bacterioopsin unfolded state.

The stability of bacteriorhodopsin (bR) has often been assessed using SDS unfolding assays that monitor the transition of folded bR (bR(f)) to unfolded (bR(u)). While many criteria suggest that the unfolding curves reflect thermodynamic stability, slow retinal (RET) hydrolysis during refolding makes it impossible to perform the most rigorous test for equilibrium, i.e.

Energetics-based discovery of protein-ligand interactions on a proteomic scale.

Biochemical functions of proteins in cells frequently involve interactions with various ligands. Proteomic methods for the identification of proteins that interact with specific ligands such as metabolites, signaling molecules, and drugs are valuable in investigating the regulatory mechanisms of cellular metabolism, annotating proteins with unknown functions, and elucidating pharmacological mechanisms. Here we report an energetics-based target identification method in which target proteins in a cell lysate are identified by exploiting the effect of ligand binding on their stabilities.

Probing membrane protein unfolding with pulse proteolysis.

Technical challenges have greatly impeded the investigation of membrane protein folding and unfolding. To develop a new tool that facilitates the study of membrane proteins, we tested pulse proteolysis as a probe for membrane protein unfolding. Pulse proteolysis is a method to monitor protein folding and unfolding, which exploits the significant difference in proteolytic susceptibility between folded and unfolded proteins.

Mapping transient partial unfolding by protein engineering and native-state proteolysis.

Transient partial unfolding of proteins under native conditions may have significant consequences in the biochemical and biophysical properties of proteins. Native-state proteolysis offers a facile way to investigate the thermodynamic and kinetic accessibilities of partially unfolded forms (cleavable forms) under native conditions. However, determination of the structure of the cleavable form, which is populated only transiently, remains challenging.

Investigating the effect of temperature on transient partial unfolding by proteolysis.

Protein inactivation frequently occurs through partially unfolded states under native conditions, and temperature is an important parameter that affects the susceptibility of proteins to inactivation. While the effect of temperature on global unfolding is well documented, however, experimental characterizations of the temperature effect on partial unfolding are rare. Proteolysis offers a valuable chance to investigate the temperature effect on partial unfolding.

Determining protein stability in cell lysates by pulse proteolysis and Western blotting.

Proteins require proper conformational energetics to fold and to function correctly. Despite the importance of having information on conformational energetics, the investigation of thermodynamic stability has been limited to proteins, which can be easily expressed and purified. Many biologically important proteins are not suitable for conventional biophysical investigation because of the difficulty of expression and purification.

Revisiting absorbance at 230nm as a protein unfolding probe.

Thermodynamic stability and unfolding kinetics of proteins are typically determined by monitoring protein unfolding with spectroscopic probes, such as circular dichroism (CD) and fluorescence. UV absorbance at 230nm (A(230)) is also known to be sensitive to protein conformation. However, its feasibility for quantitative analysis of protein energetics has not been assessed.

Investigating protein unfolding kinetics by pulse proteolysis.

Investigation of protein unfolding kinetics of proteins in crude samples may provide many exciting opportunities to study protein energetics under unconventional conditions. As an effort to develop a method with this capability, we employed "pulse proteolysis" to investigate protein unfolding kinetics. Pulse proteolysis has been shown to be an effective and facile method to determine global stability of proteins by exploiting the difference in proteolytic susceptibilities between folded and unfolded proteins.

Quantitative determination of protein stability and ligand binding by pulse proteolysis.

Pulse proteolysis exploits the difference in proteolytic susceptibility between folded and unfolded proteins for facile but quantitative determination of protein stability. The method requires only common biochemistry and molecular biology lab equipment. Pulse proteolysis also can be used to determine the affinity of a ligand to its protein target by monitoring the change in protein stability upon ligand binding.

Energetics-based protein profiling on a proteomic scale: identification of proteins resistant to proteolysis.

Native states of proteins are flexible, populating more than just the unique native conformation. The energetics and dynamics resulting from this conformational ensemble are inherently linked to protein function and regulation. Proteolytic susceptibility is one feature determined by this conformational energy landscape.