An understanding of cellular chemistry requires knowledge of how crowded environments affect proteins. The influence of crowding on protein stability arises from two phenomena, hard-core repulsions and soft (i.e., chemical) interactions. Most efforts to understand crowding effects on protein stability, however, focus on hard-core repulsions, which are inherently entropic and karacto.xyz by: On a positive note, declaring bankruptcy typically results in discharge. Discharge is when a bankruptcy court hands down a permanent order that forever prevents creditors from collecting on debts you previously incurred. Credit card debt is one common form of debt that can be discharged by a bankruptcy court. assortment of shapes and sizes (Figure 1) in its diﬀusive path. The translational diﬀusion of this substrate in such a milieu is hindered by three main factors These are: (a) crowding by large macromolecules or immobile structures that eﬀectively reduce the available volume in .
However, the influence of molecular crowding on recognition events involving virus particles, and their inhibition by antiviral compounds, is virtually unexplored. Among these processes, capsid self-assembly during viral morphogenesis and capsid-cell receptor recognition during virus entry into cells are receiving increasing attention as targets for the development of new antiviral drugs. In this study, we have analyzed the effect of macromolecular crowding on the inhibition of these two processes by peptides.
Macromolecular crowding led to a significant reduction in the inhibitory activity of: 1 , a capsid-binding peptide and a small capsid protein domain that interfere with assembly of the human immunodeficiency virus capsid, and 2 , a RGD-containing peptide able to block the interaction between foot-and-mouth disease virus and receptor molecules on the host cell membrane in this case, the effect was dependent on the conditions used.
The results, discussed in the light of macromolecular crowding theory, are relevant for a quantitative understanding of molecular recognition processes during virus infection and its inhibition. Such conditions lead to excluded volume effects arising from the mutual impenetrability of soluble macromolecules, a situation known as macromolecular crowding 1—5.
In a crowded solution, the reactivity of a given solute is determined by the number of solute molecules per unit of available not total volume.
Thus, in a molecularly crowded medium, the thermodynamic activity i. Crowding is a nonspecific force that favors reactions leading to a reduction in the total excluded volume, such as the formation of soluble macromolecular complexes or insoluble aggregates, folding of proteins, and the binding of macromolecules to surface sites.
The magnitude of the crowding effect is highly dependent on the sizes and shapes of crowding species and dilute macromolecular species. During the life cycle of viruses, most reactions in which viral macromolecules or the viral particles themselves participate also occur in molecularly crowded environments, either intracellular or extracellular.
To what extent macromolecular crowding could have an influence on the different specific recognition steps of the viral life cycle has remained, to our knowledge, a virtually unexplored question, despite its relevance for a better understanding of virus biology and the fight against viral disease.
In this study, our goal has been to experimentally analyze whether macromolecular crowding could have an influence on the inhibition by different compounds of essential steps of the viral cycle that involve virus particles. Most licensed or candidate antiviral drugs are small molecules that act by binding a viral or cellular macromolecule or macromolecular complex, such as a virus capsid, and inhibiting its function 17— In particular, two very promising antiviral strategies that are receiving increasing attention are.
For example, several experimental inhibitors of HIV-1 capsid assembly have been described 21— Some of these antiviral compounds are small peptides e. Because of their outstanding interest as new targets for antiviral therapy, in this work we have focused on these two crucial steps of the viral cycle.
As a model for the inhibition of virus capsid self-assembly, we chose HIV During HIV morphogenesis, a molecular reorganization process occurs where the mature capsid is assembled from hundreds of copies of its monomeric component, the capsid protein CA see recent reviews in 33, The CA protein is composed of two domains.
Mateu, unpublished results are also able to inhibit this process. As a model for the inhibition of virus-host cell receptor recognition we chose foot-and-mouth disease virus FMDV , the causative agent of one of the most economically important animal diseases worldwide reviewed in 41, Infection by FMDV involves the attachment of the virion to integrin receptors on the host cell membrane, through a RGD-containing protein loop the G-H loop of protein VP1 on the virus capsid reviewed in This loop is structurally free-standing and mobile, and participates in few or no contacts with other capsid regions The inhibition of FMDV infectivity by RGD peptides is due to a steric blockade of the recognition by the virion of integrin receptors on the surface of the host cells 43,47— The conceptual framework and the implications for the inhibition of crucial steps of the viral cycle are discussed.
The BHKc2 cell line used derives from a cell clone obtained by limiting dilution Solid-phase synthesized and purified peptides with an amydated C-terminus were provided by Prof. The peptides were dissolved in complete phosphate-buffered saline PBS.
The AKLH conjugate solution was dialyzed immediately before use to eliminate any free A19 peptide and other low-molecular-weight contaminants. Traces of the variation in the turbidity were analyzed as described previously The time-dependent increase in optical density fitted very well the empirical Hill function,.
The polymerization rate k p in each tested condition was determined from the slope of the approximately linear portion of the curve.
The relative amount of capsid formed was estimated from the OD corresponding to the plateau of the turbidity curves 10,35, FMD virions were purified as previously described 58 with some modifications. A quantity of 0.
Purified FMDV virions were inactivated essentially as described previously 59— Briefly, 2. Then, the suspension was dialyzed against incomplete PBS. Complete inactivation was assessed by titration in standard plaque assays. The general outline of the assay was as described previously 51 , but the conditions were appropriately varied.
The higher ratio was achieved by mixing the appropriate amounts of a suspension of infectious viral particles and a suspension of viral particles that had been completely inactivated as described above. The monolayers were fixed and stained, and the viral plaques counted. The first model system used in this study involves a competition for binding to growing HIV-1 capsidlike particles between the capsid building blocks CA dimers and other small oligomers 40 and the inhibitor molecule, either the isolated CTD or the CAI peptide.
In the absence of crowding, addition of increasing amounts of free CTD Fig. Thus both the CTD and, even more so, the CAI peptide which inhibits polymerization by a different mechanism than CTD , behaved in these experiments as effective inhibitors of HIV-1 capsid assembly in the absence of crowding. An inactive peptide used as a negative control had no inhibitory effect at all on CA polymerization data not shown.
In each panel, a representative experiment is shown. Average and standard deviation values from the processed results of several comparable experiments are shown in Fig. Each value represents the average of two, three, or four measurements obtained in independent experiments. The standard deviations are indicated. Experimental conditions were as described in the legend for Fig. Remarkably, the inhibitory activities of CTD and CAI were substantially reduced in the presence of macromolecular crowding, relative to its absence.
Similar results were obtained when the inhibition of HIV-1 capsid assembly by CAI was analyzed in the presence of other inert crowding agents including dextran T40 Fig.
As expected, the reduced inhibition under macromolecular crowding conditions was not dependent on the molecular nature of the crowding agent used. Evaluation of the cytotoxic effect of Ficoll 70 on cultured BHK21c2 cells. The values of absorbance, directly proportional to cell viability, are the average of six measurements. Next we determined the concentrations of the A15 and A19 peptides either in free form or conjugated to a large protein that were needed, in the absence of an external macromolecular crowding agent, to achieve efficient inhibition of cell attachment and infectivity of FMDV C-S8c1 Fig.
From the above experiments, a range of 0. Inhibition of FMDV infectivity by synthetic peptides. The data points for A19, A15, A15 RGE , and AKLH, respectively, represent the average of nine, four, four, or seven measurements obtained in three, two, two, or three independent experiments. The values in panels c and d , respectively, represent the average of nine or four measurements obtained in three or two independent experiments.
The ratio between the number of binding sites FMDV receptors in the membrane of infection-susceptible cells and the number of circulating viral particles may vary widely both in natural and laboratory infections. The first ratio would leave most cell receptors unoccupied by the virions, while the second ratio would be closer to saturation of the viral receptors by virions. The number of binding sites cell receptors and other experimental conditions were kept invariant.
The results of the comparison are shown in Fig. The same lack of effect of macromolecular crowding conditions on the inhibitory activity was observed for a shorter RGD peptide, A15, or for a very large AKLH peptide conjugate results not shown. The two models we have used to study the inhibition by small molecules of either capsid self-assembly or virus-cell recognition basically involve:. A large target macromolecule or macromolecular assembly T , either the growing HIV-1 capsid or the integrin receptor on the host cell membrane.
The conditional presence of an inert macromolecule C. Ficoll 70 was chosen as a model crowding agent because of its low viscosity, a size comparable to those of many proteins, and its low tendency to interact specifically with other solutes such as proteins The Ficoll concentrations used in this study would approximately correspond to the volume occupancy estimated for extracellular conditions, or only somewhat lower than typical intracellular conditions.
The use of a single inert crowding agent to approximately mimic the volume occupancy due to complex mixtures of macromolecules in biological fluids allows the assignment of the observed effects to volume exclusion. This approach avoids the complications due to additional effects that could occur if complex macromolecular mixtures were used instead see, for example, The main result we obtained using either system inhibition of capsid self-assembly or inhibition of virus-cell interactions is that macromolecular crowding can significantly reduce the inhibitory effects of the antiviral peptides tested.
Volume exclusion theory may provide a simplified conceptual framework and a general thermodynamic explanation for this experimental result: if the sizes of I competitive inhibitor and M competed macromolecule are similar, the inhibitory activity of I in a crowded solution would be similar to that in a nonphysiological, diluted ideal solution.
In contrast, if I is clearly smaller than M, the inhibitory activity of I in crowded conditions would be significantly reduced, relative to that in a dilute solution, as theoretically justified by Minton example I in 2 and summarized next.
In agreement with this theoretical prediction, it has been very recently shown that small chemical ligands that bind DNA G-quadruplexes, and thus inhibit telomerase activity, were significantly less effective under macromolecular crowding conditions However, to our knowledge the prediction that macromolecular crowding may generally impair the inhibitory activity of small compounds 2 , despite its biological relevance, had not been tested to date in any other cellular or viral system.
If the implicit assumptions and simplifications of the above theoretical analysis apply to the complex experimental models and under the conditions we have used, it could be expected that the preference ratio for association of the larger building blocks to the growing HIV-1 capsid, relative to the association of the smaller inhibitor peptides CAI and even CTD, would be higher in macromolecular crowding conditions than in a dilute solution.
Likewise, the preference ratio for association of the large FMDV virions to the cell receptors, relative to the association of the smaller inhibitor peptide A19, would be higher in macromolecular crowding conditions than in a dilute solution. For both viral models, the antiviral activity of the peptide inhibitors is predicted to be reduced in the presence of macromolecular crowding, and this is precisely what we have experimentally observed in this study.
We suggest that this may be due to an opposing, specific effect occurring in this complex system, but we are uncertain about its origin. Complexities of this system include:. The receptors were not in solution, but fixed to a cell membrane see 66 for an example regarding predictions of macromolecular crowding theory on association reactions at cellular membranes.
Because of these or other reasons, some of the simplifying assumptions made by theory may have to be revised for this complex system.
However, it is important to note that the theoretically predicted reduction in the inhibitory activity of the peptides occurred under some conditions in this system, and under any tested condition in the less complex viral capsid assembly system also investigated in this study. It is also clear that the use of a single crowding agent provides only a first approximation to experimentally study some effects of high macromolecular concentrations.
Biological environments are crowded but also heterogeneous media, and volume exclusion will not be the only factor affecting biochemical rates and equilibria. Further studies will be needed to evaluate whether the observed volume exclusion effects on virologically relevant macromolecular reactions are compounded by other effects derived from the presence of a complex mixture of macromolecules. These considerations underscore the need for more quantitative definitions of model systems and further theoretical developments to achieve a better understanding of association reactions in molecularly crowded environments.
The results obtained may also have practical implications for the initial testing of small molecules aimed at inhibiting virus assembly or virus-cell receptor recognition. This suggested approach could contribute to a better selection of candidate compounds for subsequent trials under more physiological conditions. The experimental results obtained in this study demonstrate that macromolecular crowding can, at least under certain conditions, substantially impair the inhibition by small peptide molecules of capsid self-assembly or virus attachment to receptors on the membrane of host cells.
These and other molecular recognition processes during the life cycle of viruses involve competition between ligands of widely different sizes and affinities, both outside and inside the cell. Because macromolecular crowding is widespread in both environments, volume exclusion effects may have to be considered for a more quantitative understanding of virus biology.
In addition, most antiviral drugs in use for inhibiting the activity of viral proteins, or being developed for inhibiting virus assembly or virus-cell receptor recognition, are small organic compounds.
The results obtained with two very different virus model systems indicate that the inhibitory activity of antiviral drugs may be substantially tempered under macromolecular crowding conditions such as those present in the organism. We gratefully acknowledge Prof. Minton for critical reading of the first version of the manuscript and suggestions, Prof. Andreu for the supply of peptides, and M. Fuertes for excellent technical assistance. Work in M.
Work in G. BIOC and No. National Center for Biotechnology Information , U. Journal List Biophys J v. Biophys J. Mauricio G. Author information Article notes Copyright and License information Disclaimer. Mateu: se. Received Sep 9; Accepted Dec This article has been cited by other articles in PMC.
Abstract Biological fluids contain a very high total concentration of macromolecules that leads to volume exclusion by one molecule to another. In particular, two very promising antiviral strategies that are receiving increasing attention are 1.
Synthetic peptides Solid-phase synthesized and purified peptides with an amydated C-terminus were provided by Prof. Inhibition of FMDV infectivity The general outline of the assay was as described previously 51 , but the conditions were appropriately varied. Open in a separate window. Figure 1. Figure 2. Figure 3.
Evaluation of the inhibitory activity on FMDV infectivity of RGD-containing peptides Next we determined the concentrations of the A15 and A19 peptides either in free form or conjugated to a large protein that were needed, in the absence of an external macromolecular crowding agent, to achieve efficient inhibition of cell attachment and infectivity of FMDV C-S8c1 Fig. Figure 4. Discussion The two models we have used to study the inhibition by small molecules of either capsid self-assembly or virus-cell recognition basically involve: 1.
Conclusions The experimental results obtained in this study demonstrate that macromolecular crowding can, at least under certain conditions, substantially impair the inhibition by small peptide molecules of capsid self-assembly or virus attachment to receptors on the membrane of host cells. When the transition state is more expanded than the reactant state, as in the case of protein unfolding, crowding is expected to decrease the rate constant.
The free energy of transferring the molecule X from this volume to an equal volume of solution that may be bounded by hard walls in one, two, or three dimensions is given by the statistical-thermodynamic expression Where the multiple integral in the denominator is taken over all configurational states accessible in bulk solution, and the multiple integral in the numerator is taken over all allowed configurational states in the bounded volume, that is, all states in which no part of X intersects any hard wall boundary.
While both confinement and crowding effects result from the reduction in possible configurations available to a macromolecule due to the presence of a high volume fraction of other macromolecules or static barriers to movement, there is one major qualitative difference between the two phenomena.
In contrast to the free energy cost of crowding, the free energy cost of confinement is not necessarily minimal for the molecular conformation that is globally the most compact in the sense of having the smallest radius of gyration. Rather, confinement favors conformations having a shape that is complementary to the shape of the confining volume.
For example, while a spherical conformation may be favored in a quasi-spherical cavity, the preferred conformation in a cylindrical pore may be rod-like, and the preferred conformation in a planar pore bounded by two parallel hard walls may be plate-like.
Thus numerical estimates of the magnitude of confinement effects are extremely sensitive to the choice of models for the structure of both confining space and confined macromolecular species Model calculations of the effect of confinement in differently shaped pores upon the association of two spherical monomers of identical size to form a dimer of twice the volume and varying shapes, suggest that confinement has a small effect on bimolecular association, and is expected to increase the equilibrium constant for bimolecular association by at most a factor of two or three.
However, the effect of confinement upon association constants for concerted formation of larger n -mers having a shape compatible with the shape of the confining volume increases strongly as the value of n increases If the bound ligand is completely immobile, then confinement affects only the free energy of the free ligand i. As a simple example, consider a spherical ligand with radius a confined in a spherical cavity with radius R cavity. The change in the site binding constant due to ligand confinement may be calculated from equation 2 , 4 , and 8 to be.
It is evident upon inspection of Fig 2C that the free energy cost of confining any partially or fully unfolded conformation of a protein will be greater than the cost of confining the native state, and that confinement must stabilize the native state relative to any unfolded state. As pointed out previously when summarizing the effects of crowding on protein folding, numerical estimates of the magnitude of the effect of confinement will be quite sensitive to the nature of approximations made in treating the effect of confinement on the average unfolded state.
Zhou and Dill 90 presented a simple model in which the unfolded state is modeled as a random walk with a specified radius of gyration. Some of their results are plotted in Figure 5. This result makes intuitive sense. In such small cavities it is essentially impossible for proteins to unfold, as there is no space available for proteins to unfold into. By modeling the transition state for protein folding as a sphere with a somewhat larger radius than that of the folded state, a different but conceptually related model 28 has been used to estimate the change in the energy barrier for folding, and calculate the dependence of folding rate on the size of the confining cavity.
A maximum acceleration in folding by tuning the cavity size is predicted. In conclusion, it should be noted that enhancement of association and site binding equilibria by confinement only occurs when the confined macromolecules are truly confined, i.
When protein can equilibrate between a pore and bulk solution the extent of association resulting in the formation of oligomers in the pore is less than in the bulk, and the extent of site binding in the pore equal to that in the bulk Most of the effects summarized in the preceding section can be predicted on the basis of simple statistical-thermodynamic models. These models have the advantage that they are focused on specific aspects of crowding and confinement, are intuitive, and their results can often be expressed in the form of reasonably simple analytical expressions.
On the other hand, they do not allow for the exploration of complexities associated with crowding and confinement of real macromolecules by other real macromolecules. A complementary theoretical approach is by atomistic simulations.
Zhou 94 considered the effect of crowding on the free energy of the unfolded state by treating the unfolded protein as a three-dimensional random walk in the presence of hard spherical obstacles.
Calculation of the probability that a random walk consisting of a certain number of steps will not encounter a crowding particle leads to a simple relation:. Equation 10 takes into account the possibility that an unfolded chain can in principle be accommodated within interstitial voids between spherical crowders that may be too small to accommodate a folded protein modeled as a hard particle.
When the folded protein is modeled as a hard sphere, the treatment of Zhou predicts that whereas at low volume fraction of crowder, excluded volume effects stabilize the folded state relative to the unfolded state, at very high volume fractions of crowder, excluded volume stabilizes the unfolded state relative to the folded state. Minton presented an effective two-state model for protein folding, with the unfolded state modeled as a compressible sphere.
This model also predicts the energetic consequences of neglecting intra-molecular excluded volume, and provides estimates of the extent to which the average radius of gyration of an unfolded polypeptide is reduced by addition of hard particle crowders.
Hu et al. They predicted that smaller crowders stabilize the unfolded state relative to the native state, whereas larger crowders promote the stability of the folded form. This conclusion stands in contradiction to the results of prior theoretical treatments 56 , 94 , as well as experiment In a simulation study, Cheung et al 11 found that, at a crowder volume fraction of 0. Cheung et al. The decrease in folding rate was attributed to restriction by crowders of conformational fluctuations necessary for protein folding.
A second factor may be an increase in the free energy of the transition state due to the decreased probability of finding voids that can accommodate the protein in an expanded transition state.
Using simple space-filling models for both helix and coil, Snir and Kamien 76 predicted that crowding by hard spheres promotes the formation of helical conformations by random-coil polymers.
As the folded or self-associated crowders present less excluded volume, the model predicts that the stabilization of the native state by crowders will be decreased as the crowders fold or self-associate. By modeling the unfolded state as a polymer chain and the native and transition states as hard spheres, Hayer-Hartl and Minton 28 obtained simple analytical expressions describing the dependence of the rate of two-state folding within a spherical cavity upon the radius of the cavity and the molecular weight of the confined protein.
These expressions predicted that folding rates would be maximized at an intermediate cavity size that increased with the molecular weight of the encapsulated protein. Maximization of folding rate at an intermediate cavity size has also been observed in atomistic simulations 12 , 80 , These simulations also indicated that folding rate can be modulated by attractive interactions between the confined protein and the walls of the enclosing cavity.
A simulation of Jewett et al 37 suggests that the rate of folding in a confined environment can be increased via an alternative pathway in which a folding intermediate is transiently bound to the cavity wall. In another simulation study, Rathore et al. Net stabilization is hence sequence-dependent and not as great as expected on the basis of entropic effects alone.
Ziv et al 97 simulated the helix formation of peptides confined in an infinite cylinder and found that the helical state is stabilized relative to the coil state. They attempted to rationalize their results with a simple statistical-thermodynamic model, in which the coil state is modeled as a polymer chain, and the helix is modeled by a stiffer polymer chain. Helix stabilization is predicted, but the predicted stabilization is independent of peptide length, in contrast to their simulation results.
Pande and co-workers 46 , 78 simulated helix formation inside a cylindrical pore and folding of the villin headpiece inside a spherical cavity. Unlike previous simulations, solvent water was treated explicitly in these studies.
Contrary to the result of Ziv et al 97 , confinement in the cylindrical pore was reported to disfavor helix formation. Confinement in the spherical cavity was reported to favor folding of the protein when the solvent was allowed to equilibrate with the bulk, but disfavor folding when solvent was trapped within the cavity. Elcock 18 simulated the co-translational folding of three proteins.
The proteins were fed off the peptidyltransferase active site with the ribosome large subunit, which was represented by atoms and pseudo-atoms.
No structure formation was observed while the nascent chain was in the ribosome exit tunnel, and the co-translational folding of two single-domain proteins, CI2 and barnase, were found to follow mechanisms identical to those in bulk water. However, for a two-domain protein, Semliki forest virus protein, co-translational folding was found to follow a mechanism different from that in bulk water.
In the latter environment, the two domains first folded independently and then docked together. On the ribosome, the N-terminal domain folded first and the structure of the C-terminal domain then gradually accreted onto the pre-formed domain. Relevant experimental literature published during the past four years has been classified according to one of six categories: 1 Effects of crowding on macromolecular association rates; 2 Effects of crowding on macromolecular association equilibria; 3 Effects of crowding on conformational isomerization; 4 Effects of crowding on protein stability with respect to denaturation; 5 Effects of crowding upon enzyme activity; and 6 Effects of confinement on protein stability with respect to denaturation.
Noteworthy findings in the six categories are listed in Tables 1 — 6. It has been known for some time that many partially and fully unfolded proteins exhibit an increased propensity in vitro to form insoluble aggregates, leading to irreversible denaturation 1 , 13 , These two types of stability are in fact ordinarily interdependent and may be treated separately only under special conditions, such as in the limit of extreme dilution.
The close relationship between the two types of stability is due to the similarity on an atomic scale between the kinds of non-covalent interactions that stabilize the native conformation of a protein and those that stabilize intermolecular non-covalent complexes. The effect of macromolecular crowding on a particular reaction is generally studied experimentally by measuring changes in reaction rates or equilibria in the presence of different concentrations of putatively inert macromolecular cosolutes.
One of the most popular cosolutes is the highly water-soluble synthetic polymer polyethylene glycol or polyethylene oxide , which is actually a polyether with monomeric structure —CH 2 — CH 2 — O —. It has long been evident that PEG fractions with molecular weights in excess of a few thousand have a large and predominantly repulsive interaction with proteins, and tend to induce macromolecular associations and compaction in qualitative accord with crowding theory see for example 36 , 67 and works cited in Tables 1 — 5.
However, a number of studies have shown that this interaction cannot be described quantitatively in terms of excluded volume alone, and several independent lines of evidence point to an attractive interaction between PEG and nonpolar or hydrophobic sidechains on the protein surface 5 , 6 , 50 , 83 , Thus the repulsive excluded volume contribution to PEG-protein interaction is partially compensated to an unknown extent by an attractive interaction, the strength of which can vary significantly between different proteins of approximately equal size.
A variety of other water-soluble polymers and proteins e. Dextrans, Ficoll, hemoglobin, defatted BSA have been shown to lack such an attractive interaction for other proteins, and their interactions with proteins can be described using pure excluded volume models 42 , 50 , 69 , 70 , 73 , Since these readily available polysaccharides and proteins have the added advantage of resembling more closely the types of macromolecules encountered in a physiological medium, we recommend them as alternatives to PEG as crowding agents for use in quantitative studies.
Theoretical models for estimating the magnitude of crowding and confinement on macromolecular reactions generally assume that these effects are predominantly entropic in origin, i. Nevertheless, it has been realized from the outset that other nonspecific interactions such as electrostatic repulsion and attraction and hydrophobic attraction are likely to contribute significantly to overall energetics in highly crowded or confined media It is assumed in these models that crowder molecules interact with each other only via volume exclusion.
One indication that the situation in physiological media may be more complex is provided by a recently published report 87 , where concentrated mixtures of protein and polysaccharide were shown to exhibit non-additive effects upon the refolding of lysozyme.
Moreover, concentrated solution mixtures of dextran and polyethylene glycol have been found to spontaneously separate into immiscible phases, between which proteins may partition in accordance with their relative affinity for each phase The physical bases of these phenomena deserve closer study, as one would expect the local environment of most biological macromolecules in vivo to consist of more than one volume-excluding species.
We cannot yet adequately assess functional consequences of the nearly ubiquitous proximity of soluble proteins to the surfaces of biological structures e. However some directions for future research have been suggested by relevant in vitro studies. A variety of proteins have been shown to associate weakly with actin fibers, microtubules, DNA, and phospholipid membranes in a non site-specific fashion see 10 for references to specific studies.
The reduction in configurational entropy of the protein resulting from this dual mode of localization is greater than that achieved by hard wall confinement alone, and in fact magnifies significantly the consequences of confinement.
Simple theoretical models 53 predict that adsorbed macromolecules, like hard-wall confined macromolecules, have a stronger tendency to self- or hetero-associate than they do in bulk solution, and that the tendency to associate increases substantially with the strength of attraction between the soluble macromolecule and the surface.
Adsorption may also increase the rate of macromolecular binding to specific surface sites A number of macromolecular associations have been reported to proceed more rapidly or to a greater extent on surfaces than in bulk solution e.
Experimental techniques have been developed recently that provide information about either the stability and conformation or the association properties of specific labeled proteins within living cells reviewed in The use of some of these techniques to study diffusional transport of labeled macromolecules in cytoplasm and tissue is reviewed elsewhere in this volume. In these studies a labeled protein or pair of labeled proteins is introduced into a cell via expression of recombinant proteins or microinjection.
Then, a signal that reflects conformation or association of labeled protein, or co-localization hetero-association of two labeled proteins is monitored. In certain experiments, the intracellular environment is globally perturbed via addition of denaturant, temperature change, or application of hyper- or hypo-osmotic stress 21 , 32 , 33 , The monitored response of the labeled protein s within the cell to the applied perturbation is compared to that of the same protein s in dilute solution, and conclusions are drawn regarding the effect of the environment in vivo upon the stability of the labeled protein s.
While each of these techniques does indeed provide information about aspects of the behavior of tracer proteins within a cell, one must be very careful about the interpretation of the results of such experiments. A number of additional questions must be answered: 1 How is the test protein distributed among the different local microenvironments within an intact cell? If it is found in multiple environments, how does one interpret the overall average signal? Does this response alter the distribution of the test protein, the composition of the microenvironment s of the test protein, and the interactions between the test protein and its surroundings within each microenvironment?
Such a bottom-up approach would ultimately lead to construction of a cytomimetic medium incorporating all of the major elements thought to be present in the selected microenvironment.
The ability to control and independently manipulate temperature, pH, salt and osmolyte concentration, the types and concentrations of soluble bystander macromolecules and the types and abundances of structural elements such as membranes and cytoskeletal filaments if appropriate in this model system provides a rigorous approach to the characterization of nonspecific interactions influencing the behavior of proteins and other macromolecules within a native-like environment.
Clearly this is no simple task, but we believe that if our goal is to understand in quantitative terms the role of nonspecific interactions in biology — a role we believe is absolutely essential to the mechanism of life — we cannot avoid paying attention to the details of these inherently complex systems [ Footnote 5 ]. Macromolecular crowding nonspecifically enhances reactions leading to the reduction of total excluded volume.
In general these include the formation of macromolecular complexes in solution, binding of macromolecules to surface sites, formation of insoluble aggregates, and compaction or folding of proteins. The expected magnitude of the effect is strongly dependent upon the relative sizes and shapes of concentrated crowding species and dilute macromolecular reactants and products. Macromolecular crowding is generally expected to increase the rate of slow, transition-state limited association reactions and to decrease the rate of fast, diffusion-limited association reactions.
Simple statistical-thermodynamic theories based upon coarse-grained structural models usually provide reliable predictions of qualitative effects, and in favorable circumstances can provide reasonably good semi-quantitative predictions of the magnitude of an expected effect. Biological fluids are more complex than systems studied theoretically or experimentally in vitro due to increased heterogeneity and the probable presence of nonspecific attractive and repulsive intermolecular interactions in addition to volume exclusion.
Theoretical and experimental exploration of model systems containing well-defined elements of added complexity are strongly encouraged. Reported effects of macromolecular crowding on protein stability with respect to denaturation. Zimmerman, pioneering investigator of excluded volume effects in biological systems, with emphasis on the influence of macromolecular crowding on the structure and function of DNA. However, differences between Helmholtz and Gibbs free energy changes associated with reactions in the liquid state are not of qualitative significance.
National Center for Biotechnology Information , U. Annu Rev Biophys. Author manuscript; available in PMC Feb Minton 3. Allen P. Author information Copyright and License information Disclaimer. Huan-Xiang Zhou: ude. Minton: vog. Copyright notice. The publisher's final edited version of this article is available at Annu Rev Biophys. See other articles in PMC that cite the published article.
Abstract Expected and observed effects of volume exclusion on the free energy of rigid and flexible macromolecules in crowded and confined systems, and consequent effects of crowding and confinement on macromolecular reaction rates and equilibria are summarized. Keywords: excluded volume, configurational entropy, free energy, protein-protein interactions, macromolecular associations, protein folding, site-binding. Introduction It is now widely appreciated that almost all proteins and other biological macromolecules in vivo exist, at least transiently, as components of structural and functional complexes 3.
Open in a separate window. Figure 1. Figure 2. Association equilibria Fig 1A Consider a bimolecular association reaction I taking place in a solution of spherical crowding molecules. Figure 3. Figure 4. Two-state protein folding equilibria Fig 1C The presence of crowder will influence equilibria between conformational states of a macromolecule in favor of conformations that exclude less volume to crowder.
Association equilibria Fig. Site binding equilibria Fig. Two-state protein folding equilibria Fig 2C It is evident upon inspection of Fig 2C that the free energy cost of confining any partially or fully unfolded conformation of a protein will be greater than the cost of confining the native state, and that confinement must stabilize the native state relative to any unfolded state.
Figure 5. Effects of crowding on protein folding Zhou 94 considered the effect of crowding on the free energy of the unfolded state by treating the unfolded protein as a three-dimensional random walk in the presence of hard spherical obstacles. Effect of crowding on macromolecular isomerization Using simple space-filling models for both helix and coil, Snir and Kamien 76 predicted that crowding by hard spheres promotes the formation of helical conformations by random-coil polymers.
Effects of confinement on isomerization and protein folding By modeling the unfolded state as a polymer chain and the native and transition states as hard spheres, Hayer-Hartl and Minton 28 obtained simple analytical expressions describing the dependence of the rate of two-state folding within a spherical cavity upon the radius of the cavity and the molecular weight of the confined protein.
Miscellaneous confinement effects Elcock 18 simulated the co-translational folding of three proteins. Review of the experimental literature since Relevant experimental literature published during the past four years has been classified according to one of six categories: 1 Effects of crowding on macromolecular association rates; 2 Effects of crowding on macromolecular association equilibria; 3 Effects of crowding on conformational isomerization; 4 Effects of crowding on protein stability with respect to denaturation; 5 Effects of crowding upon enzyme activity; and 6 Effects of confinement on protein stability with respect to denaturation.
Table 1 Reported effects of macromolecular crowding on association rates. Addition of PEG 3. In the absence of the scaffolding protein, the capsid protein forms amorphous polymers in PEG. At low crowder concentration, rate scales with rotational diffusion. At higher concentrations, rate decreases more strongly with increasing polymer concentration, possibly due to crowding-induced self-association of reactant species.
Table 6 Reported effects of macromolecular confinement on protein stability and conformation. Confinement in polyacrylamide gels increases T m of several proteins 7. T m increases with increasing polyacrylamide concentration in gel. Confinement of glucose isomerase in functionalized mesoporous silica enhances retention of specific activity at high urea concentrations Refolding is accelerated in larger cavities and decelerated in smaller cavities, in qualitative or semi-quantitative agreement with predictions of confinement simulations and models 28 , 39 , Effect of crowding upon the competition between protein folding and aggregation It has been known for some time that many partially and fully unfolded proteins exhibit an increased propensity in vitro to form insoluble aggregates, leading to irreversible denaturation 1 , 13 , A cautionary note on the use of polyethylene glycol PEG as a crowding agent The effect of macromolecular crowding on a particular reaction is generally studied experimentally by measuring changes in reaction rates or equilibria in the presence of different concentrations of putatively inert macromolecular cosolutes.
Table 5 Reported effects of macromolecular crowding on enzyme activity. Enzymatic activity increases, then decreases with increasing concentration of protein crowding agents, but decreases monotonically with increasing concentration of polymeric crowding agents Addition of dextrans 15 — K or Ficoll 70K reduces rate of hydrolysis catalyzed by alkaline phosphatase Effect attributed largely to reduction in rate of enzyme-substrate encounter.
Specific activity of hexokinase reduced in high concentrations of BSA Reaction rates measured calorimetrically Effects of small osmolytes and high concentrations of BSA on hexokinase activity are additive Consistent with hypothesis that osmolytes except urea interact primarily with proteins via volume exclusion Addition of PEG 4 — 20K enhances activity of DNase I and S1 nuclease, does not significantly affect activity of exonuclease III, and decreases activity of exonuclease I From in vitro to in vivo : Addressing additional complications in biological environments Beyond excluded volume: effects of other types of nonspecific interactions Theoretical models for estimating the magnitude of crowding and confinement on macromolecular reactions generally assume that these effects are predominantly entropic in origin, i.
Interpretation and significance of in-cell measurements of protein stability and association Experimental techniques have been developed recently that provide information about either the stability and conformation or the association properties of specific labeled proteins within living cells reviewed in Summary points Macromolecular crowding nonspecifically enhances reactions leading to the reduction of total excluded volume.
Addition of the unrelated protein RNase A promotes the dimerization of tracer apomyoglobin In contrast, an equivalent mass concentration of HSA did not promote apoMb dimer formation; this differential is much larger than expected from excluded volume models.
Addition of dextran 10 K enhances formation of a decamer of bovine pancreatic trypsin inhibitor Addition of dextran 70 K inhibits the exchange of subunits between aggregates of alpha-crystallin.
Table 3 Reported effects of macromolecular crowding on conformational isomerization. Addition of dextran reduces mean distance between residues and in partially unfolded adenylate kinase Table 4 Reported effects of macromolecular crowding on protein stability with respect to denaturation.
Addition of Ficoll 70K increases the free energy of unfolding of FKbinding protein Two-state transition verified. Magnitude consistent with prediction of excluded volume model Addition of dextran 30K stabilizes the molten globule conformation of apomyoglobin at pH 2 with respect to heat and cold-induced unfolding Results in qualitative agreement with prediction of excluded volume theory. Unfolding rate little affected.
Zimmerman, pioneering investigator of excluded volume effects in biological systems, with emphasis on the influence of macromolecular crowding on the structure and function of DNA 1 Equations 1 — 3 apply equally to changes in Helmholtz or Gibbs free energies.
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