The structure of a protein complex, together with information about its affinity and other thermodynamic characteristics, provide a “frozen” view of the complex. This picture ignores the kinetic nature of protein-protein association and dissociation, which are of major biological and biophysical interest. This review focuses on recent advances in deciphering the kinetic pathway of protein complex formation, the nature of the pre-complex formed through diffusion (which we have termed the “transient complex”1), the transition state, and other intermediates (such as the so-called encounter complex) along the association pathway.

Protein-protein association is at the center of diverse biological processes ranging from enzyme catalysis/inhibition to regulation of immune response by cytokines. The association rates often play a critical role in such processes, as in situations where speed is of essence.2 For example, the purple cone snail and other venomous animals capture prey with remarkable efficiency and speed by releasing toxins that rapidly bind to ion channels;3 the green mamba achieves a similar feat by targeting acetylcholinesterase (AChE), an enzyme essential for the integrity of neural transmission.4 Bacteria such as Escherichia coli and Bacillus amyloliquefaciens excrete nucleases as weapons against competitors or predators. Defense of the producing cells from damage to their own DNA or RNA by such nucleases requires rapid association with cognate inhibitors.5,6 Indeed, in the last example rapid association is such a priority that the inhibitor barstar has a cluster of acidic residues that facilitate association with the nuclease barnase, even though the clustered charges reduce folding stability.7 In the ruminant gut, RNase A is required for degrading accumulated RNA; potential toxicity of leaked nuclease is prevented by rapid association with a ribonuclease inhibitor.8,9

Reorganization of the actin cytoskeleton provides yet another illustration of the importance of rapid protein association. Reorganization is attained through actin polymerization, which is nucleated by the Arp2/3 complex. The latter is activated by the Wiskott-Aldrich Syndrome protein (WASp), which in turn is released from the auto-inhibited state by the Rho GTPase Cdc42.10 As actin polymerization is initiated with a nucleation process, the speed of upstream signaling has a critical impact on the rate of polymer formation. It is thus not surprising that high association rate constants have been observed between partners along the signaling pathway.11,12 The high association rate constant between Cdc42 and WASp has been found to be essential for the latter to stimulate actin polymerization, as another Rho GTPase sharing 70% sequence identity, TC10, with an identical dissociation rate constant but a 1000-fold lower association rate constant, failed to stimulate actin polymerization.11 The failure to stimulate actin polymerization in patients carrying mutant WAS genes is the root cause of the Wiskott-Aldrich Syndrome.

Several other compelling arguments can be made for the biological roles of rapid protein association.13 (a) Fast association may enhance binding affinity. High affinity can also be achieved through slow dissociation; however, for proteins involved in signaling, slow dissociation is not an option, since it implies a long-lasting bound state, which effectively corresponds to a permanent off- or on-switch. A good example for this is the binding of Ras to its natural affector Raf. This protein dissociates within a fraction of a second, but maintains an affinity in the nM range through fast association. Moreover, the difference between the natural effector, Raf, and the non-natural effector, Ral, lies in their rates of association with Ras.14 Therefore, even if not for a direct reason (such as in stimulation of actin polymerization), the affinity requirement alone may call for fast association. (b) Enzyme-substrate binding is a determining factor for the overall turnover rate and becomes the rate-limiting step for catalytically “perfect” enzymes. Substrate-binding rate constants of such enzymes reach 108 M−1s−1 and beyond, as found for the ribotoxin restrictocin and RNase A.15,16 (c) When several proteins compete for the same receptor or when one protein is faced with alternative pathways, kinetic control, not thermodynamic control, dominates in many cases; this is especially true when dissociation is slow. For example, during protein synthesis cognate and noncognate aminoacyl-tRNA synthetases can potentially compete for the same tRNA. As an additional example, consider newly synthesized proteins, which potentially face aggregation if not isolated by a chaperone. From the point of view of kinetic control, it is easy to see why rapid binding of denatured proteins to the chaperonin GroEL has been observed.17 (d) Differences in binding rate between related proteins may serve as an additional mechanism for specificity, as can be suggested for Rho GTPases Cdc42 and TC10 and for Ras effectors Raf and Ral.

The examples and arguments presented above suggest that rapid binding is as important as high affinity in the proper functioning of proteins. It is now increasingly recognized that proteins function in the context of multi-component complexes. Manipulating association rate constants of various components presents unique opportunities for the control of protein functions. Many interactions between proteins are also targeted for drug development; in designing such drugs, both high affinity and rapid binding should be taken into consideration.

1.1. Overview of Protein Association Kinetics
The observed rate constants of protein association span a wide range, from 109 M−1s−1 (Figure 1). In comprehending these values, a basic fact is that, for two proteins to recognize each other, their interfaces have to be oriented with high specificity. A relative rotation of as little as a few degrees or a relative translation by a few Angstroms is sufficient to break all specific interactions between the two proteins.18 The rate of association of a protein complex is limited by diffusion and geometric constraints of the binding sites, and may be further reduced by subsequent chemical processes.19






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Figure 1


The wide spectrum of association rate constants. The red vertical line marks the start of the diffusion-controlled regime. The shaded range marks the absence of long-range forces. Adapted with permission from Ref. 1. Copyright 2008 Wiley Interscience..

/pdf/fundamental-aspects-of-protein-protein-association-kinetics-3z35bnbxbs.pdf

Fundamental aspects of protein-protein association kinetics.

In this paper, centrifuge-based microfluidic platforms are reviewed and compared with other popular microfluidic propulsion methods. The underlying physical principles of centrifugal pumping in microfluidic systems are presented and the various centrifuge fluidic functions, such as valving, decanting, calibration, mixing, metering, heating, sample splitting, and separation, are introduced. Those fluidic functions have been combined with analytical measurement techniques, such as optical imaging, absorbance, and fluorescence spectroscopy and mass spectrometry, to make the centrifugal platform a powerful solution for medical and clinical diagnostics and high throughput screening (HTS) in drug discovery. Applications of a compact disc (CD)-based centrifuge platform analyzed in this review include two-point calibration of an optode-based ion sensor, an automated immunoassay platform, multiple parallel screening assays, and cellular-based assays. The use of modified commercial CD drives for high-resolution optical imaging is discussed as well. From a broader perspective, we compare technical barriers involved in applying microfluidics for sensing and diagnostic use and applying such techniques to HTS. The latter poses less challenges and explains why HTS products based on a CD fluidic platform are already commercially available, whereas we might have to wait longer to see commercial CD-based diagnostics.

https://www.annualreviews.org/doi/pdf/10.1146/annurev.bioeng.8.061505.095758

Lab on a cd

https://www.cell.com/biophysj/pdf/S0006-3495(95)80095-0.pdf

The biophysics of DNA hybridization with immobilized oligonucleotide probes.

http://nano.mae.cornell.edu/erickson_AnalBiochem_DNAhybridization.pdf

Modeling of DNA hybridization kinetics for spatially resolved biochips

This review article presents an overview of some of the tools, techniques and applications of numerical simulation for integrated microfluidic devices. Provided is a broad overview of the different areas to which numerical techniques have been applied in the development of these devices from detailed studies of fundamental microfluidic problems (e.g., species mixing and sample dispersion) to unique approaches that take a more global overview of the entire system. While the majority of the work to date has been in these areas, also reviewed is some recent progress into other equally important areas of microscale transport such as thermal analysis and chemical reactivity and specificity. An overview of the advantages and disadvantages of common numerical techniques is also presented along with a brief discussion of some of the existing numerical tools, focusing on those best suited for microscale transport analysis. As microfluidic devices become increasingly complex, optimal fluidic and transport designs become more and more difficult to do experimentally. Thus, it is believed that future demand in the field will be for highly integrated simulation tools that allow users without a significant computational fluids background to “numerical prototype” highly integrated devices.

/pdf/towards-numerical-prototyping-of-labs-on-chip-modeling-for-53l076v17e.pdf

Towards numerical prototyping of labs-on-chip: modeling for integrated microfluidic devices

Reduction-of-dimensionality kinetics at reaction-limited cell surface receptors.

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Reduction-of-dimensionality kinetics at reaction-limited cell surface receptors.