Gilad Haran

Fundamental Aspects of Protein-Protein Association Kinetics

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 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.. ... To better understand the kinetics of association of two proteins (A and B), it is useful to consider the process as going through an intermediate state (A*B), in which the two proteins have near-native separations and orientations.1,20–23 We refer to this intermediate state as the transient complex,1,20 noting that is sometimes also termed the encounter complex.24 A more detailed discussion of terminology, as well as the specification of the ensemble of configurations making up the transient complex is provided in Section 3. From this ensemble, conformational rearrangement can lead to the native complex (C). Accordingly we have the kinetic scheme A+B⇄k−DkDA∗B→kcC (1) While the first step of this scheme depends on relative diffusion between the protein molecules, the second step is akin to an intramolecular chemical reaction, and can therefore be described by the classical transition-state theory25 (with the transition state located at the top of the free energy barrier separating A*B from C26) or by Kramers’ theory.27 The latter theory accounts for barrier recrossing and models motion along the reaction coordinate as diffusive. The overall rate constant of association is ka=kDkck−D+kc (2) which is bounded by the diffusion-controlled rate constant, kD, for reaching the transient complex. This limit is reached when conformational rearrangement is fast relative to the dissociation of the transient complex (i.e., kc ≫ k−D), leading to

Watching proteins fold one molecule at a time

Recent theoretical work suggests that protein folding involves an ensemble of pathways on a rugged energy landscape. We provide direct evidence for heterogeneous folding pathways from single-molecule studies, facilitated by a recently developed immobilization technique. Individual fluorophore-labeled molecules of the protein adenylate kinase were trapped within surface-tethered lipid vesicles, thereby allowing spatial restriction without inducing any spurious interactions with the environment, which often occur when using direct surface-linking techniques. The conformational fluctuations of these protein molecules, prepared at the thermodynamic midtransition point, were studied by using fluorescence resonance energy transfer between two specifically attached labels. Folding and unfolding transitions appeared in experimental time traces as correlated steps in donor and acceptor fluorescence intensity. The size of the steps, in fluorescence resonance energy transfer efficiency units, shows a very broad distribution. This distribution peaks at a relatively low value, indicating a preference for small-step motion on the energy landscape. The time scale of the transitions is also distributed, and although many transitions are too fast to be time-resolved here, the slowest ones may take >1 sec to complete. These extremely slow changes during the folding of single molecules highlight the possible importance of correlated, non-Markovian conformational dynamics.

Managing light polarization via plasmon–molecule interactions within an asymmetric metal nanoparticle trimer

The interaction of light with metal nanoparticles leads to novel phenomena mediated by surface plasmon excitations. In this article we use single molecules to characterize the interaction of surface plasmons with light, and show that such interaction can strongly modulate the polarization of the emitted light. The simplest nanostructures that enable such polarization modulation are asymmetric silver nanocrystal trimers, where individual Raman scattering molecules are located in the gap between two of the nanoparticles. The third particle breaks the dipolar symmetry of the two-particle junction, generating a wavelength-dependent polarization pattern. Indeed, the scattered light becomes elliptically polarized and its intensity pattern is rotated in the presence of the third particle. We use a combination of spectroscopic observations on single molecules, scanning electron microscope imaging, and generalized Mie theory calculations to provide a full picture of the effect of particles on the polarization of the emitted light. Furthermore, our theoretical analysis allows us to show that the observed phenomenon is very sensitive to the size of the trimer particles and their relative position, suggesting future means for precise control of light polarization on the nanoscale.

Role of Solvation Effects in Protein Denaturation: From Thermodynamics to Single Molecules and Back

Protein stability often is studied in vitro through the use of urea and guanidinium chloride, chemical cosolvents that disrupt protein native structure. Much controversy still surrounds the underlying mechanism by which these molecules denature proteins. Here we review current thinking on various aspects of chemical denaturation. We begin by discussing classic models of protein folding and how the effects of denaturants may fit into this picture through their modulation of the collapse, or coil-globule transition, which typically precedes folding. Subsequently, we examine recent molecular dynamics simulations that have shed new light on the possible microscopic origins of the solvation effects brought on by denaturants. It seems likely that both denaturants operate by facilitating solvation of hydrophobic regions of proteins. Finally, we present recent single-molecule fluorescence studies of denatured proteins, the analysis of which corroborates the role of denaturants in shifting the equilibrium of the coil-globule transition.

Effects of denaturants and osmolytes on proteins are accurately predicted by the molecular transfer model

Interactions between denaturants and proteins are commonly used to probe the structures of the denatured state ensemble and their stabilities. Osmolytes, a class of small intracellular organic molecules found in all taxa, also profoundly affect the equilibrium properties of proteins. We introduce the molecular transfer model, which combines simulations in the absence of denaturants or osmolytes, and Tanfords transfer model to predict the dependence of equilibrium properties of proteins at finite concentration of osmolytes. The calculated changes in the thermodynamic quantities (probability of being in the native basin of attraction, m values, FRET efficiency, and structures of the denatured state ensemble) with GdmCl concentration [C] for the protein L and cold shock protein CspTm compare well with experiments. The radii of gyration of the subpopulation of unfolded molecules for both proteins decrease (i.e., they undergo a collapse transition) as [C] decreases. Although global folding is cooperative, residual secondary structures persist at high denaturant concentrations. The temperature dependence of the specific heat shows that the folding temperature (TF) changes linearly as urea and trimethylamine N-oxide (TMAO) concentrations increase. The increase in TF in TMAO can be as large as 20°C, whereas urea decreases TF by as much as 35°C. The stabilities of protein L and CspTm also increase linearly with the concentration of osmolytes (proline, sorbitol, sucrose, TMAO, and sarcosine).

Trimeric Plasmonic Molecules: The Role of Symmetry

Artificial plasmonic molecules possess excitation modes that are defined by their symmetry and obey group theory rules, just like conventional molecules. We follow the evolution of surface-plasmon spectra of plasmonic trimers, assembled from equal-sized silver nanoparticles, as gradual geometric changes break their symmetry. The spectral modes of an equilateral triangle, the most symmetric structure of a trimer, are degenerate. This degeneracy is lifted as the symmetry is lowered when one of the vertex angles in opened, which also leads to a subtle transition between bright and dark modes. Our experimental results are quantitatively explained using numerical simulations and plasmon hybridization theory.

Multiple-Particle Nanoantennas for Enormous Enhancement and Polarization Control of Light Emission

We investigate the light emission from dipolar emitters located within nanoparticle antennas. It is found that the enormous emission enhancement can reach nearly a million fold. For multinanoparticle antennas, the polarization of the emissions strongly depends on the geometry of the antennas, the emitted wavelengths, and the dielectric functions of surrounding media. It is shown that a polarization nanorotator, which modulates the emission polarization on the nanometer scale, can be readily realized by varying either the geometry or surrounding media of nanoparticle antennas.

How, when and why proteins collapse: the relation to folding

Unfolded proteins under strongly denaturing conditions are highly expanded. However, when the conditions are more close to native, an unfolded protein may collapse to a compact globular structure distinct from the folded state. This transition is akin to the coil-globule transition of homopolymers. Single-molecule FRET experiments have been particularly conducive in revealing the collapsed state under conditions of coexistence with the folded state. The collapse can be even more readily observed in natively unfolded proteins. Time-resolved studies, using FRET and small-angle scattering, have shown that the collapse transition is a very fast event, probably occurring on the submicrosecond time scale. The forces driving collapse are likely to involve both hydrophobic and backbone interactions. The loss of configurational entropy during collapse makes the unfolded state less stable compared to the folded state, thus facilitating folding.

Single-molecule fluorescence spectroscopy maps the folding landscape of a large protein

Proteins attain their function only after folding into a highly organized three-dimensional structure. Much remains to be learned about the mechanisms of folding of large multidomain proteins, which may populate metastable intermediate states on their energy landscapes. Here we introduce a novel method, based on high-throughput single-molecule fluorescence experiments, which is specifically geared towards tracing the dynamics of folding in the presence of a plethora of intermediates. We employ this method to characterize the folding reaction of a three-domain protein, adenylate kinase. Using thousands of single-molecule trajectories and hidden Markov modelling, we identify six metastable states on adenylate kinases folding landscape. Remarkably, the connectivity of the intermediates depends on denaturant concentration; at low concentration, multiple intersecting folding pathways co-exist. We anticipate that the methodology introduced here will find broad applicability in the study of folding of large proteins, and will provide a more realistic scenario of their conformational dynamics.

Plasmonic Control of the Shape of the Raman Spectrum of a Single Molecule in a Silver Nanoparticle Dimer

We study surface-enhanced Raman scattering (SERS) of individual organic molecules embedded in dimers of two metal nanoparticles. The good control of the dimer preparation process, based on the usage of bifunctional molecules, enables us to study quantitatively the effect of the nanoparticle size on the SERS intensity and spectrum at the single molecule level. We find that as the nanoparticle size increases the total Raman intensity increases and the lower energy Raman modes become dominant. We perform an electromagnetic calculation of the Raman enhancement and show that this behavior can be understood in terms of the overlap between the plasmonic modes of the dimer structure and the Raman spectrum. As the nanoparticle size increases, the plasmonic dipolar mode shifts to longer wavelength and thereby its overlap with the Raman spectrum changes. This suggests that the dimer structure can provide an external control of the emission properties of a single molecule. Indeed, clear and systematic differences are observed between Raman spectra of individual molecules adsorbed on small versus large particles.