Exploring the charge space of protein-protein association: a proteomic study

The rate of association of a protein complex is a function of an intrinsic basal rate and of the magnitude of electrostatic steering. In the present study we analyze the contribution of electrostatics towards the association rate of proteins in a database of 68 transient hetero-protein-protein complexes. Our calculations are based on an upgraded version of the computer algorithm PARE, which was shown to successfully predict the impact of mutations on k(on) by calculating the difference in Columbic energy of interaction of a pair of proteins. HyPare (http://bip.weizmann.ac.il/HyPare), automatically calculates the impact of mutations on a per-residue basis for all residues of a protein-protein interaction, achieving a precision similar to that of PARE. Our calculations show that electrostatics play a marginal role (<10 fold) in determining the rate of association for about half of the complexes in the database. Strong electrostatic steering, which results in an increase of over 100-fold in k(on), was calculated for about 25% of the complexes. Applying HyPare to all 68 complexes in the database shows that a small number of residues are hotspots for association. About 40% of the hotspots are calculated to increase the rate of association upon mutation, and thus increase binding affinity. This is a much higher ratio than found for hotspots for dissociation, where the large majority cause weaker binding. About 40% of the hotspots are located outside the physical boundary of the binding site, making them ideal candidates for protein engineering. Our data shows that a majority of protein-protein complexes are not optimized for fast association. Hotspots are not evenly distributed between all types of amino acids. About 75% of all hotspots are of charged residues. This is understandable, as a charge-reverse mutant changes the total charge by 2. The small number of hydrophobic residues that are hotspots upon mutation probably relates to their location and surrounding. For 18 out of the 68 complexes in the database, experimental values of k(on) are available. For these, a basal rate of association was calculated to be in the range of 10(4)M(-1)s(-1) to 10(7)M(-1)s(-1). Some of these rates were verified independently from experimental mutant data. The basal rates were correlated with the size of the proteins and the shape of the interface.     

In vivo dynamics of Rac-membrane interactions

The small GTPase Rac cycles between the membrane and the cytosol as it is activated by nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). Solubility in the cytosol is conferred by binding of Rac to guanine-nucleotide dissociation inhibitors (GDIs). To analyze the in vivo dynamics of Rac, we developed a photobleaching method to measure the dissociation rate constant (k(off)) of membrane-bound GFP-Rac. We find that k(off) is 0.048 s(-1) for wtRac and approximately 10-fold less (0.004 s(-1)) for G12VRac. Thus, the major route for dissociation is conversion of membrane-bound GTP-Rac to GDP-Rac; however, dissociation of GTP-Rac occurs at a detectable rate. Overexpression of the GEF Tiam1 unexpectedly decreased k(off) for wtRac, most likely by converting membrane-bound GDP-Rac back to GTP-Rac. Both overexpression and small hairpin RNA-mediated suppression of RhoGDI strongly affected the amount of membrane-bound Rac but surprisingly had only slight effects on k(off). These results indicate that RhoGDI controls Rac function mainly through effects on activation and/or membrane association.     

Measuring dissociation rate constants of protein complexes through subunit exchange: experimental design and theoretical modeling

Protein complexes are dynamic macromolecules that constantly dissociate into, and simultaneously are assembled from, free subunits. Dissociation rate constants, k(off), provide structural and functional information on protein complexes. However, because all existing methods for measuring k(off) require high-quality purification and specific modifications of protein complexes, dissociation kinetics has only been studied for a small set of model complexes. Here, we propose a new method, called Metabolically-labeled Affinity-tagged Subunit Exchange (MASE), to measure k(off) using metabolic stable isotope labeling, affinity purification and mass spectrometry. MASE is based on a subunit exchange process between an unlabeled affinity-tagged variant and a metabolically-labeled untagged variant of a complex. The subunit exchange process was modeled theoretically for a heterodimeric complex. The results showed that k(off) determines, and hence can be estimated from, the observed rate of subunit exchange. This study provided the theoretical foundation for future experiments that can validate and apply the MASE method.     

Prediction of protein-protein association rates from a transition-state theory

We recently developed a theory for the rates of protein-protein association. The theory is based on the concept of a transition state, which separates the bound state, with numerous short-range interactions but restricted translational and rotational freedom, and the unbound state, with, at most, a small number of interactions but expanded configurational freedom. When not accompanied by large-scale conformational changes, protein-protein association becomes diffusion limited. The association rate is then predicted as k(a)=k(a)(0)exp(-DeltaG(el)(double dagger)/k(B)T), where DeltaG(el)(double dagger) is the electrostatic interaction free energy in the transition state, k(a)(0) is the rate in the absence of electrostatic interactions, and k(B)T is thermal energy. Here, this transition-state theory is used to predict the association rates of four protein complexes. The predictions for the wild-type complexes and 23 mutants are found to agree closely with experimental data over wide ranges of ionic strength.     

Predicting the rate enhancement of protein complex formation from the electrostatic energy of interaction

The rate of association of proteins is dictated by diffusion, but can be enhanced by favorable electrostatic forces. Here the relationship between the electrostatic energy of interaction, and the kinetics of protein-complex formation was analyzed for the protein pairs of: hirudin-thrombin, acetylcholinesterase-fasciculin and barnase-barstar, and for a panel of point mutants of these proteins. Electrostatic energies of interaction were calculated as the difference between the electrostatic energy of the complex and the sum of the energies of the two individual proteins, using the computer simulation package DelPhi. Calculated electrostatic energies of interaction were compared to experimentally determined rates of association. One kcal/mol of Coulombic interaction energy increased the rate of association by a factor of 2.8, independent of the protein-complex or mutant analyzed. Electrostatic energies of interaction were also determined from the salt dependence of the association rate constant, using the same basic equation as for the theoretical calculation. A Br?nsted analysis of the electrostatic energies of interactions plotted versus experimentally determined ln(rate)s of association shows a linear relation between the two, with a beta value close to 1. This is interpreted as the energy of the transition state varies according to the electrostatic interaction energy, fitting a two state model for the association reaction. Calculating electrostatic rate enhancement from the electrostatic interaction energy can be used as a powerful tool to design protein complexes with altered rates of association and affinities.     

Electrostatic rate enhancement and transient complex of protein-protein association

The association of two proteins is bounded by the rate at which they, via diffusion, find each other while in appropriate relative orientations. Orientational constraints restrict this rate to approximately 10(5)-10(6) M(-1) s(-1). Proteins with higher association rates generally have complementary electrostatic surfaces; proteins with lower association rates generally are slowed down by conformational changes upon complex formation. Previous studies (Zhou, Biophys J 1997;73:2441-2445) have shown that electrostatic enhancement of the diffusion-limited association rate can be accurately modeled by $k_{\bf D}$ = $k_{D}0\ {exp} ( - \langle U_{el} \rangle;{\star}/k_{B} T),$ where k(D) and k(D0) are the rates in the presence and absence of electrostatic interactions, respectively, U(el) is the average electrostatic interaction energy in a "transient-complex" ensemble, and k(B)T is the thermal energy. The transient-complex ensemble separates the bound state from the unbound state. Predictions of the transient-complex theory on four protein complexes were found to agree well with the experiment when the electrostatic interaction energy was calculated with the linearized Poisson-Boltzmann (PB) equation (Alsallaq and Zhou, Structure 2007;15:215-224). Here we show that the agreement is further improved when the nonlinear PB equation is used. These predictions are obtained with the dielectric boundary defined as the protein van der Waals surface. When the dielectric boundary is instead specified as the molecular surface, electrostatic interactions in the transient complex become repulsive and are thus predicted to retard association. Together these results demonstrate that the transient-complex theory is predictive of electrostatic rate enhancement and can help parameterize PB calculations.     

Kinetic and structural studies of specific protein-protein interactions in substrate catalysis by Cdc25B phosphatase

Using a combination of steady-state and single-turnover kinetics, we probe substrate association, dissociation, and chemistry for the reaction of Cdc25B phosphatase with its Cdk2-pTpY/CycA protein substrate. The rate constant for substrate association for the wild-type enzyme is 1.3 x 10(6) M(-1) s(-1). The rate constant for dissociation is slow compared to the rate constant for phosphate transfer to form the phospho-enzyme intermediate (k2 = 1.1 s(-1)), making Cdk2-pTpY/CycA a sticky substrate. Compared to the wild type, all hotspot mutants of residues at the remote docking site that specifically affect catalysis with the protein substrate (Arg488, Arg492, and Tyr497 on Cdc25B and Asp206 on Cdk2) have greatly slowed rate constants of association (70- to 4500-fold), and some mutants have decreased k2 values compared to that of the wild type. Most dramatically, R492L, despite showing no significant changes in a crystal structure at 2.0 A resolution, has an approximately 100-fold decrease in k2 compared to that of wild-type Cdc25B. The active site C473S mutant binds tightly to and dissociates slowly from Cdk2-pTpY/CycA (Kd = 10 nM, k(off) = 0.01 s(-1)). In contrast, the C473D mutant, despite showing only localized perturbations in the active site at 1.6 A resolution, has a much weaker affinity and dissociates rapidly (Kd of 2 microM, k(off) > 2 s(-1)) from the protein substrate. Overall, we demonstrate that the association of Cdc25B with its Cdk2-pTpY/CycA substrate is governed to a significant extent by the interactions of the remote hotspot residues, whereas dissociation is governed by interactions at the active site.     

Electrostatic recognition of matrix targeting signal by mitochondrial processing peptidase

Mitochondrial processing peptidase (MPP), a metalloendopeptidase consisting of alpha and beta subunits, specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off the N-terminal basic matrix targeting signals (MTS). Basic residues in MTS and acidic sites in MPP are required for effective processing. To elucidate whether the enzyme recognizes the MTS through electrostatic interaction, we investigated the effects of various salts on MPP activity. Decreases in the activity depended on the ionic strength and increases in the Michaelis constant value correlated clearly with the ionic strength, indicating a lower affinity of the enzyme for the substrate. Direct determination of the affinity between MPP and a MTS peptide using surface plasmon resonance showed a decrease in the association rate with high salt and that dissociation constant values were decreased. The effect of salt on the processing activity towards a variety of precursors was confirmed using five precursors with different sequences and lengths of MTS. Thus, we propose that electrostatic interactions are indispensable for the association between various MTS and MPP.     

Linkage of EcoRI dissociation from its specific DNA recognition site to water activity, salt concentration, and pH: separating their roles in specific and non-specific binding.

We have measured the dependencies of both the dissociation rate of specifically bound EcoRI endonuclease and the ratio of non-specific and specific association constants on water activity, salt concentration, and pH in order to distinguish the contributions of these solution components to specific and non-specific binding. For proteins such as EcoRI that locate their specific recognition site efficiently by diffusing along non-specific DNA, the specific site dissociation rate can be separated into two steps: an equilibrium between non-specific and specific binding of the enzyme to DNA, and the dissociation of non-specifically bound protein. We demonstrated previously that the osmotic dependence of the dissociation rate is dominated by the equilibrium between specific and non-specific binding that is independent of the osmolyte nature. The remaining osmotic sensitivity linked to the dissociation of non-specifically bound protein depends significantly on the particular osmolyte used, indicating a change in solute-accessible surface area. In contrast, the dissociation of non-specifically bound enzyme accounts for almost all the pH and salt-dependencies. We observed virtually no pH-dependence of the equilibrium between specific and non-specific binding measured by the competition assay. The observed weak salt-sensitivity of the ratio of specific and non-specific association constants is consistent with an osmotic, rather than electrostatic, action. The seeming lack of a dependence on viscosity suggests the rate-limiting step in dissociation of non-specifically bound protein is a discrete conformational change rather than a general diffusion of the protein away from the DNA.     

NMR study of volatile anesthetic binding to nicotinic acetylcholine receptors

New lines of evidence suggest that volatile anesthetics interact specifically with proteins. Direct binding analysis, however, has been largely limited to soluble proteins. In this study, specific interaction was investigated between isoflurane, a clinically important volatile anesthetic, and membrane-bound nicotinic acetylcholine receptors (nAChRs) from Torpedo electroplax, using (19)F nuclear magnetic resonance spectroscopy and gas chromatography. The receptors were reconstituted into 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid vesicles. After correcting for nonspecific partitioning into the lipid, the equilibrium dissociation constant, K(d), of isoflurane binding to nAChR at 15 degrees C was found to be 0.36 +/- 0.03 mM. This value is within the clinically relevant concentration range of the agent. Based on the receptor concentrations in the vesicle suspension assayed by the bicinchoninic acid method and the fraction of bound isoflurane, X(b), determined by gas chromatography, an estimate of an average of 9-10 specifically bound isoflurane molecules can be made for each receptor, or two for each subunit. Upon binding, the transverse relaxation time constant (T(2)) of (19)F resonance of isoflurane is decreased by nearly three orders of magnitude, indicating a dramatic reduction in the mobility of specifically bound isoflurane. Kinetic analysis reveals that the off rate of binding, k(-1), is 1.7 x 10(4) s(-1). The on rate, k(+1), can thus be calculated to be approximately 4.8 x 10(7) M(-1) s(-1), suggesting a nearly diffusion-limited association. This is in contrast to anesthetic binding to a soluble protein, bovine serum albumin (BSA), where k(+1) and k(-1) are at least an order of magnitude slower. It is concluded that the presence of lipids may be critical for the correct evaluation of binding kinetics between volatile anesthetics and neuronal receptors.     

Kinetic analysis of acetylation-dependent Pb1 bromodomain-histone interactions

Stopped-flow fluorescence anisotropy was used to determine the kinetic parameters that define acetylation-dependent bromodomain-histone interactions. Bromodomains are acetyllysine binding motifs found in many chromatin associated proteins. Individual bromodomains were derived from the polybromo-1 protein, which is a subunit of the PBAF chromatin-remodeling complex that has six tandem bromodomains in the amino-terminal region. The average k(on) and k(off) values for the formation of high-affinity complexes are 275 M(-1) s(-1) and 0.41 x 10(-3) s(-1), respectively. The average k(on) and k(off) values for the formation of low-affinity complexes are 119 M(-1) s(-1) and 1.42 x 10(-3) s(-1), respectively. Analysis of the on- and off-rates yields acetylation site-dependent equilibrium dissociation constants averaging 1.4 and 12.9 microM for high- and low-affinity complexes, respectively. This work represents the first examination of kinetic mechanisms of acetylation-dependent bromodomain-histone interactions.     

Protein translocation through Tom40: kinetics of peptide release

Mitochondrial proteins are almost exclusively imported into mitochondria from the cytosol in an unfolded or partially folded conformation. Regardless of whether they are destined for the outer or inner membrane, the intermembrane space, or the matrix, proteins begin the importation process by crossing the mitochondrial outer membrane via a specialized protein import machinery whose main component is the Tom40 channel. High-resolution ion conductance measurements through the Tom40 channel in the presence of the mitochondrial presequence peptide pF(1)β revealed the kinetics of peptide binding. Here we show that the rates for association k(on) and dissociation k(off) strongly depend on the applied transmembrane voltage. Both kinetic constants increase with an increase in the applied voltage. The increase of k(off) with voltage provides strong evidence of peptide translocation. This allows us to distinguish quantitatively between substrate blocking and permeation.     

Kinetic analysis of the interaction of the C1 domain of protein kinase C with lipid membranes by stopped-flow spectroscopy

The diacylglycerol (DG)/phorbol ester-dependent translocation of conventional protein kinase C (PKC) isozymes is mediated by the C1 domain, a membrane-targeting module that also selectively binds phosphatidylserine (PS). Using stopped-flow spectroscopy, we dissect the contribution of DG/phorbol esters (C1 ligand) and PS in driving the association and dissociation of the C1 domain from membranes. Specifically, we examine the binding to membranes of the C1B domain of PKCbeta with a substituted Trp (Y123W) whose fluorescence is quenched upon binding to membranes. Binding of this construct (C1Bbeta-Y123W) to phospholipid vesicles is cooperative with respect to PS content and dependent on C1 ligand, as previously characterized. Stopped-flow analysis reveals that the apparent association rate (k(on)(app)), but not the apparent dissociation rate (k(off)(app)), is highly sensitive to PS content: the 60-fold increase in membrane affinity for vesicles containing no PS compared with 40 mol % PS results primarily from a robust (30-fold) increase in k(on)(app) with little effect (2-fold) on k(off)(app). Membrane affinity is also controlled by the content and structure of the C1 ligand. In contrast to PS, these ligands markedly alter k(off)(app) with smaller effects on k(on)(app). We also show that the affinity for phorbol ester-containing membranes is 2 orders of magnitude higher than that for DG-containing membranes primarily resulting from differences in k(off)(app). Our data are consistent with a model in which the C1 domain is recruited to the membrane via an initial weak electrostatic interaction with PS, followed by a rapid two-dimensional search for ligand, the binding of which retains the domain at the membrane. Thus, PS drives the initial encounter, and DG/phorbol esters retain the domain on membranes. The decreased effectiveness of DG compared with phorbol esters in retaining the C1 domain on membranes contributes to the molecular dichotomy of the rapid, transient nature of DG-dependent PKC signaling versus the chronic hyperactivity of phorbol ester-activated PKC.     

Sequence specific recognition of ssDNA by a lupus autoantibody: kinetics and mechanism of binding.

11F8 is a pathogenic anti-ssDNA monoclonal autoantibody isolated from a lupus-prone mouse. Previous studies have established that 11F8 is sequence specific. To determine the basis for the observed binding specificity, stopped-flow fluorescence spectroscopy was used to measure the kinetic parameters and establish the mechanisms for the association of 11F8 with its target sequence, noncognate, and nonspecific ssDNA ligands. The data revealed that sequence-specific binding follows a two-step mechanism where the initial association step is second order. Values of k(1) are fast and above the modified Smoluchowski limit for a diffusion limited interaction (10(5)-10(6)M(-1)s(-1)). The dependency of k(1) on [salt] and solvent polarity indicates that electrostatic steering is responsible for this rapid association rate. The second association step is rate limiting and is characteristic of an isomerization process during which binding interfaces are optimized. This step apparently is driven by the desolvation of hydrophobic surfaces within the binding interface. The differences in the rate of dissociation for the various DNA ligands suggest that specificity is governed primarily through the dissociation of the final complexes.     

Kinetic and thermodynamic framework for assembly of the six-component bI3 group I intron ribonucleoprotein catalyst

The yeast mitochondrial bI3 group I intron RNA splices in vitro as a six-component ribonucleoprotein complex with the bI3 maturase and Mrs1 proteins. We report a comprehensive framework for assembly of the catalytically active bI3 ribonucleoprotein. (1) In the absence of Mg(2+), two Mrs1 dimers bind independently to the bI3 RNA. The ratio of dissociation to association rate constants, k(off)/k(on), is approximately equal to the observed equilibrium K(1/2) of 0.12 nM. (2) At magnesium ion concentrations optimal for splicing (20 mM), two Mrs1 dimers bind with strong cooperativity to the bI3 RNA. k(off)/k(on) is 15-fold lower than the observed K(1/2) of 11 nM, which reflects formation of an obligate intermediate involving one Mrs1 dimer and the RNA in cooperative assembly of the Mrs1-RNA complex. (3) The bI3 maturase monomer binds to the bI3 RNA at almost the diffusion-controlled limit and dissociates with a half-life of 1 h. k(off)/k(on) is approximately equal to the equilibrium K(D) of 2.8 pM. The bI3 maturase thus represents a rare example of a group I intron protein cofactor whose binding is adequately characterized by a one-step mechanism under conditions that promote splicing. (4) Maturase and Mrs1 proteins each bind the bI3 RNA tightly, but with only modest coupling (approximately 1 kcal/mol), suggesting that the proteins interact at independent RNA binding sites. Maturase binding functions to slow dissociation of Mrs1; whereas prior Mrs1 binding increases the bI3 maturase k(on) right to the diffusion limit. (5) At effective concentrations plausibly present in yeast mitochondria, a predominant assembly pathway emerges involving rapid, tight binding by the bI3 maturase, followed by slower, cooperative assembly of two Mrs1 dimers. In the absence of other factors, disassembly of all protein subunits will occur in a single apparent step, governed by dissociation of the bI3 maturase.     

Membrane binding kinetics of protein kinase C betaII mediated by the C2 domain.

Conventional isoforms of protein kinase C (PKC) are activated when their two membrane-targeting modules, the C1 and C2 domains, bind the second messengers diacylglycerol (DG) and Ca2+, respectively. This study investigates the mechanism of Ca2+-induced binding of PKC betaII to anionic membranes mediated by the C2 domain. Stopped-flow fluorescence spectroscopy reveals that Ca2+-induced binding of the isolated C2 domain to anionic vesicles proceeds via at least two steps: (1) rapid binding of two or more Ca2+ ions to the free domain with relatively low affinity and (2) diffusion-controlled association of the Ca2+-occupied domain with vesicles. Ca2+ increases the affinity of the C2 domain for anionic membranes by both decreasing the dissociation rate constant (k(off)) and increasing the association rate constant (k(on)) for membrane binding. For binding to vesicles containing 40 mol % anionic lipid in the presence of 200 microM Ca2+, k(off) and k(on) are 8.9 s(-1) and 1.2 x 10(10) M(-1) x s(-1), respectively. The k(off) value increases to 150 s(-1) when free Ca2+ levels are rapidly reduced, decreasing the average lifetime of the membrane-bound C2 domain (tau = k(off)(-1)) from 110 ms in the presence of Ca2+ to 6.7 ms when Ca2+ is rapidly removed. Experiments addressing the role of electrostatic interactions reveal that they stabilize either the initial C2 domain-membrane encounter complex or the high-affinity membrane-bound complex. Specifically, lowering the phosphatidylserine mole fraction or including MgCl2 in the binding reaction decreases the affinity of the C2 domain for anionic vesicles by both reducing k(on) and increasing k(off) measured in the presence of 200 microM Ca2+. These species do not affect the k(off) value when Ca2+ is rapidly removed. Studies with PKC betaII reveal that Ca2+-induced binding to membranes by the full-length protein proceeds minimally via two kinetically resolvable steps: (1) a rapid bimolecular association of the enzyme with vesicles near the diffusion-controlled limit and, most likely, (2) subsequent conformational changes of the membrane-bound enzyme. As is the case for the C2 domain, k(off) for full-length PKC betaII increases when Ca2+ is rapidly removed, reducing tau from 11 s in the presence of Ca2+ to 48 ms in its absence. Thus, both the C2 domain and the slow conformational change prolong the lifetime of the PKC betaII-membrane ternary complex in the presence of Ca2+, with rapid membrane release triggered by removal of Ca2+. These results provide a molecular basis for cofactor regulation of PKC whereby the C2 domain searches three-dimensional space at the diffusion-controlled limit to target PKC to relatively common anionic phospholipids, whereupon a two-dimensional search is initiated by the C1 domain for the more rare, membrane-partitioned DG.     

Energetics of Shaker K channels block by inactivation peptides

A synthetic peptide of the NH2-terminal inactivation domain of the ShB channel blocks Shaker channels which have an NH2-terminal deletion and mimics many of the characteristics of the intramolecular inactivation reaction. To investigate the role of electrostatic interactions in both peptide block and the inactivation process we measured the kinetics of block of macroscopic currents recorded from the intact ShB channel, and from ShB delta 6-46 channels in the presence of peptides, at different ionic strengths. The rate of inactivation and the association rate constants (k(on)) for the ShB peptides decreased with increasing ionic strength. k(on) for a more positively charged peptide was more steeply dependent on ionic strength consistent with a simple electrostatic mechanism of enhanced diffusion. This suggests that a rate limiting step in the inactivation process is the diffusion of the NH2-terminal domain towards the pore. The dissociation rates (k(off)) were insensitive to ionic strength. The temperature dependence of k(on) for the ShB peptide was very high, (Q10 = 5.0 +/- 0.58), whereas k(off) was relatively temperature insensitive (Q10 approximately 1.1). The results suggest that at higher temperatures the proportion of time either the peptide or channel spends in the correct conformation for binding is increased. There were two components to the time course of recovery from block by the ShB peptide, indicating two distinct blocked states, one of which has similar kinetics and dependence on external K+ concentration as the inactivated state of ShB. The other is voltage- dependent and at -120 mV is very unstable. Increasing the net charge on the peptide did not increase sensitivity to knock-off by external K+. We propose that the free peptide, having fewer constraints than the tethered NH2-terminal domain binds to a similar site on the channel in at least two different conformations.     

Effect of crowding on protein-protein association rates: fundamental differences between low and high mass crowding agents

Physiological media constitutes a crowded environment that serves as the field of action for protein-protein interaction in vivo. Measuring protein-protein interaction in crowded solutions can mimic this environment. In this work we follow the process of protein-protein association and its rate constants (k(on)) of the beta-lactamase (TEM)-beta-lactamase inhibitor protein (BLIP) complex in crowded solution using both low and high molecular mass crowding agents. In all crowded solutions (0-40% (w/w) of ethylene glycol (EG), poly(ethylene glycol) (PEG) 200, 1000, 3350, 8000 Da Ficoll-70 and Haemaccel the measured absolute k(on), but not k(off) values, were found to be slower as compared to buffer. However, there is a fundamental difference between low and high mass crowding agents. In the presence of low mass crowding agents and Haemaccel k(on) depends inversely on the solution viscosity. In high mass polymer solutions k(on) changes only slightly, even at viscosities 12-fold higher than water. The border between low and high molecular mass polymers is sharp and is dictated by the ratio between the polymer length (L) and its persistence length (Lp). Polymers that are long enough to form a flexible coil (L/Lp > 2) behave as high molecular mass polymers and those who are unable to do so (L/Lp < 2) behave as low molecular mass polymers. We concluded that although polymers solution are crowded, this property is not uniform; i.e. there are areas in the solution that contain bulk water, and in these areas proteins can diffuse and associate almost as if they were in diluted environment. This porous medium may be taken as mimicking some aspects of the cellular environment, where many of the macromolecules are organized along membranes and the cytoskeleton. To determine the contribution of electrostatic attraction between proteins in crowded milieu, we followed k(on) of wt-TEM and three BLIP analogs with up to 100-fold increased values of k(on) due to electrostatic steering. Faster associating BLIP variants keep their relative advantage in all crowded solutions, including Haemaccel. This result suggests that faster associating protein complexes keep their advantage also in complex environment.