Macromolecular crowding in vitro, in vivo, and in\nbetween
Biochemical processes take place in heterogeneous and highly volume occupied or crowded environments that can considerably influence the reactivity and distribution of participating macromolecules. Here we summarize the thermodynamic consequences of excluded volume and longer-ranged nonspecific intermolecular interactions for macromolecular reactions in volume-occupied media. In addition, we summarize and compare the information content of studies of crowding in vitro and in vivo. We emphasize the importance of characterizing the behavior not only of labeled tracer macromolecules, but also the composition and behavior of unlabeled macromolecules in the immediate vicinity of the tracer. Finally, we propose strategies for extending quantitative analyses of crowding in simple model systems to increasingly complex media up to and including intact cells.
What is macromolecular crowding?<!>What have we learned from theory and in vitro experiment?<!>What have we learned from \xe2\x80\x9cin vivo\xe2\x80\x9d experiments?<!>How can we connect bottom-up (in vitro) and top-down (in vivo)\nobservations?<!>Concluding Remarks and Future Perspectives
<p>The term "macromolecular crowding" was originally used to
connote the influence of a high total weight per volume concentration of
macromolecules on the equilibria and kinetics of macromolecular reactions taking
place in a highly volume-occupied or "crowded" solution, independent
of whether any single species of reactant or product was concentrated1. The observation that almost every
biological fluid medium, both extracellular and intracellular, contains a
substantial total concentration of macromolecules has led to a widespread hope and
expectation that an understanding of the physical-chemical origins of crowding
effects will help us to understand how biochemical reactions within a living
organism may differ, and by how much, from those observed in a test tube2–5. It is however necessary to recognize that all differences
between specified biochemical equilibria and rates within an intact cell and the
same equilibria and rates in a dilute solution of purified macromolecules cannot and
should not be attributed to crowding. One cannot ignore the likelihood of additional
modulating effects deriving from interactions of soluble macromolecules with
membranes and other structural elements6–9, in addition
to unidentified or incompletely characterized interactions between the studied
reactants and other soluble species such as molecular chaperones10. Thus we would like to make clear
at the outset that the term macromolecular crowding should be used to describe only
those effects arising from the presence of a total high weight per volume
concentration of functionally unrelated soluble macromolecules.</p><p>The influence of crowding on macromolecular equilibria has traditionally been
studied through the use of simple theoretical and experimental models that may be
explored in quantitative detail. The motivation for this approach has been the idea
that only when simple systems are well understood, should one attempt to add and
study theoretical and/or experimental elements of additional complexity in a
stepwise fashion, with the goal of eventually reaching an understanding of systems
that mimic physiological media. This approach has been described as
"bottom-up"11. In contrast, a growing number of recent publications describe
attempts to characterize the effects of crowding on macromolecular equilibria and
kinetics directly within intact cells. One might describe such an approach as
"top-down", although the term must be qualified, as discussed
subsequently. The purpose of this critical review is to compare the information
content of the two approaches and to suggest how future progress may be optimized by
a combination of both.</p><!><p>The basic thermodynamic relations governing the effects of crowding on
macromolecular equilibria are summarized in Box 1. The effects of crowding on equilibria, kinetics and transport
properties have been more completely described elsewhere12–16.
It follows from the relations presented in Box
1 that in order to understand the physical basis of an observed crowding
effect, information about the free energies of transfer of reactants and products
from a dilute to a crowded medium is essential.</p><p>The free energy of transfer is essentially equal to the free energy of
interaction between a molecule of the newly introduced species (termed probe) and
all of the other macromolecules (termed crowders) at equilibrium in the crowded
medium17. The free energy
of transfer depends upon the effective potential of interaction between the probe
and crowder molecules in solution, the nature of which may be deduced from the
composition dependence of various solution properties (see for example18–21).</p><p>From the results of many in vitro experiments conducted upon purified
macromolecules under precisely specified conditions we have learned that the total
free energy of interaction between a particular probe or trace species at low
concentration and a particular crowding species at arbitrarily high concentration is
the sum of several contributions. The most basic and often dominant contribution,
called volume exclusion, is an entropic effect arising from the mutual
impenetrability of macromolecules (Figure 1).
The effects of excluded volume upon transfer free energies become evident only when
the fraction of total volume occupied by macromolecules becomes substantial and when
the size of the probe species is comparable to or greater than the size of the
concentrated or crowder species13.
The contribution of other longer-ranged interactions to free energies of transfer
and chemical equilibria12 will be
discussed subsequently.</p><p>Early theoretical treatments of the effect of "crowding" or
high total macromolecular volume occupancy upon macromolecular reactions focused
upon universal contribution of volume exclusion arising from mutual impenetrability
of reactants, products and crowding species to the free energies of
transfer22–24. This is because steric repulsion
is undeniably the most universal form of nonspecific interaction25. Simple models for estimating the
free energies of transfer of macromolecules from a dilute to a crowded solution were
based upon the recognition that the effective potential acting between compact
globular proteins in solution could be approximated by a hard particle potential
acting between equivalent convex particles (i.e., no interaction beyond contact
distances)12.
Well-developed theories of convex hard particle fluids26–28
were then employed to estimate the free energy associated with the introduction of a
tracer species into a fluid consisting of one or more crowder species at arbitrary
concentration. Oosawa and Asakura had earlier theoretically predicted that a high
concentration of macromolecules would induce an effective attraction between larger
colloidal particles29, 30, and their results are equivalent
to those of excluded volume theory in the limit of low crowder concentration.
However, the formalism of Oosawa and Asakura does not provide a convenient
quantitative description of the effect of excluded volume on chemical reactions and
so far has not been utilized for that purpose.</p><p>Experimental studies carried out on many model systems designed to reduce the
effect of nonspecific interactions other than volume exclusion have shown that the
hard particle model provides a powerful qualitative, semi-quantitative and often
quantitative means for prediction and analysis of crowding effects in these systems.
Earlier studies have been reviewed in13–15, and
recent studies include31–35. At the same time, experimental
measurement of the pH dependence of colligative properties of protein solutions
revealed that under other conditions, longer-ranged repulsive and attractive
electrostatic interactions between macromolecules contribute significantly to the
chemical potential, and hence reactivity, of macromolecules in concentrated
solutions36, 37. Nonspecific electrostatic
attraction between probe and crowder species was found to counteract the effects of
volume exclusion to a varying extent, and in certain instances to dominate the
overall behavior of individual tracer proteins, peptides, and nucleotides19, 38–40. Several
approaches to the generalization of crowding theory to allow for enthalpic as well
as entropic intermolecular interactions have been published21, 36, 38, 41–46.
While these generalized theories can be used to rationalize experimental results,
they are not yet capable of quantitatively predicting the effect of a specific
macromolecular crowding species upon a specific macromolecular reaction.</p><p>Finally, preliminary in vitro studies indicate that the effects of mixed
crowders (protein + polymer, polymer + DNA, protein + protein,) on the stability and
aggregation propensity of tracer proteins may be far from additive of the effects of
each individual crowder47–49. Thus the study of crowding has
become more complex and experimentally challenging as we recognize that the
nonspecific intermolecular interactions underlying crowding effects depend not only
upon the abundances and gross sizes and shapes of probe and crowder species, but
also upon their chemical compositions and environmental variables.</p><p>In Box 2 we list a number of
conditions that should be satisfied if the results of any crowding experiment
conducted in vitro are to be amenable to quantitative analysis. We believe that this
should serve as a checklist when such experiments are designed.</p><!><p>Among life scientists, the chief motivation for studying the properties of
crowded media is to understand how crowding may impact macromolecular reactions in
physiological media. Estimates of the total concentration of macromolecules
(proteins, nucleic acids, polysaccharides) in a cell range from 200 to over 400
g/l50–52, but these estimates are extremely
rough and do not distinguish between mobile or diffusible species and immobile
species incorporated into static structural elements. The general strategy employed
by investigators studying "in cell crowding" is to introduce
fluorescently or isotopically labeled tracer proteins into the cell by means of
transfection, microinjection, or recombinant expression. The kinetics and/or
equilibria of reactions involving the tracer species are then characterized by
fluorescence microscopy or NMR spectroscopy53–59.</p><p>Although the intact cell presents an attractive system for the study of
"crowding in vivo", the interpretation of results obtained from such
studies must take into account complexity and heterogeneity of the cellular
interior. Even the simplest of prokaryotic organisms provides a variety of
microenvironments that are sufficiently distinct in composition (Figure 2) so that a single type of probe molecule
within each of these microenvironments may exhibit significantly different transport
and thermodynamic properties, which may derive not only from bulk crowding, but
confinement60,
adsorption6 and
liquid-liquid phase separation61, 62 as well. The interior of a
eukaryotic cell is far more complex, containing a variety of membrane-enclosed
organelles, as well as a network of cytoskeletal fibers. The relative contributions
of crowding, confinement, and adsorption to macromolecular reactivity will likely
differ between each of the intracellular microenvironments.</p><p>In the original "in cell" experiments aimed at
characterizing the stability of a particular protein, the labeled probe molecule was
introduced into the cell, but no information was available regarding the
distribution of the probe among microenvironments that may differ widely in
composition. The signal obtained from the labeled probe (or probes) was thus treated
as a whole cell average. Such treatment is problematic for at least two reasons. (1)
If the probe is limited to a single microenvironment, the signal observed reflects
only the behavior of the probe in that unidentified microenvironment. (2) If the
probe is distributed between multiple microenvironments, the cell average signal may
not reflect the behavior of the probe in any of the individual microenvironments, or
may reflect the behavior of the probe primarily in the microcompartments into which
it preferentially distributes.</p><p>Technical advances in fluorescence microscopy have provided the ability to
measure a probe signal as a function of location within an intact cell55, 56, 63. Experiments
conducted using this technique have shown clearly that the equilibrium thermal
stability and kinetics of conformational change of a given fluorescently labeled
probe can vary substantially within a single cell64, 65, and
between cells of the same type39.
For example, the positional variability of folding kinetics and equilibrium of a
mutant of phosphoglycerokinase fused to green and red fluorescent proteins in intact
U2OS (mammalian) cells is clearly non-random (Figures
3A,B), and appears to correlate with supramolecular structures such as
membranes or cytoskeletal fibers within the cell55. This could indicate that the in-cell behavior of a probe
molecule may be influenced by its interaction with surfaces as much as by crowding
in the bulk medium, as has been suggested previously6, 8, 66, 67.</p><p>Although fluorescence microscopy has provided enhanced spatial resolution,
the origin of the observed heterogeneous behavior remains indeterminate, since the
positional and temporal variations in the local composition of unlabeled
macromolecules and their states of association remain largely unknown. In addition,
when measuring thermal stability of a fluorescent probe within the cell, one cannot
neglect the possibility of changes in the association state and abundance of
unlabeled crowding macromolecules that may be either incidental to or associated
with a homeostatic response of the living cell to changes in temperature. Finally,
recent studies have shown that the results of in-cell experiments are subject to
qualitative variation with the choice of fluorescent label68 and choice of probe molecule69, 70.</p><p>Further complexity becomes evident when one considers the possibility that
total macromolecular concentration, a strong modulator of the magnitude of the
crowding effect, may vary with location within the cell, since osmolarity may be
maintained by compensatory variation in electrolyte concentration. Even within
regions of roughly constant total macromolecular concentration, a local
microenvironment is likely to contain multiple crowding species, some of which may
have a net repulsive interaction with the probe, and some of which may have a net
attractive interaction with the probe. It follows that if the probe is free to
distribute itself within the cellular interior, then at equilibrium it will tend to
accumulate preferentially in the most attractive (oppositely charged) and/or the
least crowded microenvironments. If this is the case, the probe will not report
crowding effects in the most volume-excluded and/or like-charged microenvironments,
where the consequences of crowding are likely to be greatest.</p><p>Measurements of fluorescence resonance energy transfer (FRET) and
fluorescence correlation and cross-correlation have also been used, to characterize
the kinetics and equilibria of binary heteroassociation of pairs of proteins bearing
donor and acceptor labels58, 59. In addition, FRET-based
"crowding sensors" consisting of donor-acceptor pairs linked by a
flexible polymer56 or a
polypeptide consisting of two helical regions linked by a flexible
"hinge"54
have been designed. It is expected that the intensity of observed FRET, which is a
function of the average distance between the donor and acceptor, will be a measure
of the degree to which a particular crowded environment promotes or inhibits
association of the donor/acceptor pair (contraction of the polymer chain or closing
of the "hinge").</p><p>Many of the complications and uncertainties associated with the
interpretation of in cell measurements of thermal stability discussed earlier in
this section apply to the interpretation of the results of these studies of
association as well. In one study of hetero-association of probe molecules in intact
cells, the authors reported "significant differences in protein mobility and
Kd [dissociation equilibrium constant]" for the identical
association reaction examined in two different cell types. The latter result is not
at all surprising since it is well established that variations in the size,
abundance and chemical identity of crowding species have a major influence upon the
magnitude of the crowding effect on protein association43, 48, 49. Thus conclusions drawn from a
study of the behavior of a single set of probe reactants in a single cell, even if
correct for the particular system studied, cannot be assumed to apply to a different
reaction in a different microenvironment within the same cell, another cell of the
same type, or in another cell type.</p><!><p>The above considerations do not invalidate the potential value of in-cell
measurements. However, one must keep in mind that such experiments in and of
themselves do not constitute a "top-down" analysis, since they are
only the first step in such an analysis. Future progress depends upon moving from
the top down and from the bottom up.</p><p>First, methods should be developed that will permit fractionation of cell
contents in a way that preserves as well as possible each of the various
microenvironments. Such fractionation is probably more readily accomplished with
eukaryotic cells containing discrete organelles71, but even in E. coli the nucleoid can be separated from the
proteinaceous cytoplasm, and the remaining soluble proteins and macromolecular
complexes separated from membranous environments72.</p><p>Second, chemical and/or proteomic analysis of the contents of a nominally
homogeneous microcompartment should be employed to provide information about the
identity and abundance of major macromolecular crowder species, and biophysical
techniques73, 74 should be employed to ascertain
their states of association in situ.</p><p>Third, control experiments should be conducted to ensure that probe labels
do not introduce artifactual interactions between probe and the environment. The
behavior of different probe species and differences in probe labeling, including
exchange of the position of donor and acceptor fluorophores used in a FRET
measurement68, 75, 76, should be characterized within a single uniform
microenvironment in order to determine whether probe-independent conclusions may be
drawn from the experimental results.</p><p>Fourth, the equilibrium behavior of the selected probe or probes should be
characterized within individual isolated microcompartments. We do not believe that
whole cell lysate is a satisfactory substitute for fractionated organelles, since
the process of lysis artificially homogenizes cell contents, mixing macromolecules
that are not present together in intact cells.</p><p>Fifth, the behavior of the selected probe or probes should then be
characterized in the presence of individual macromolecular constituents of the
microcompartment at their "in-vivo" abundances and well-defined
mixtures of these constituents. These studies should include the influence on probe
behavior of probe-accessible membranes and cytoskeletal fibers if present8, 77. Attempts should be made to determine the minimal composition
of the partially reconstituted "semi-synthetic" microenvironment in
which the behavior of the probe is comparable or identical to that observed in the
intact microenvironment?</p><p>Sixth, attention should be paid to the presence and abundance of molecules
like small heat shock proteins within a particular environment, that preferentially
bind to partially unfolded proteins10,
78 and could thus promote
overall destabilization of a probe.</p><p>And finally, in a medium as complex as the interior of an intact cell it is
absolutely necessary to distinguish modulation of biochemical reactions arising from
macromolecular crowding from other modulating effects such as the nonspecific
interaction of proteins with membranes or cytoskeletal structures8, 79, and the partitioning of reactants between immiscible
phases80.</p><!><p>To date, most measurements of probe protein behavior within cells have been
aimed at characterizing the stability and dynamics of the probe molecule. The
results of such experiments are in many cases not susceptible to quantitative
interpretation due to our lack of knowledge of the structure of the unfolded state
(or manifold of unfolded states) of the probe protein, and how structure and
transfer free energy of the unfolded state are affected by both excluded volume and
attractive interactions with the crowded environment. Moreover, the global unfolding
and refolding of proteins within cells is likely controlled by strong interactions
with molecular chaperones and accessory proteins rather than by thermodynamic
equilibrium10, 81, so the biological relevance of
studies of thermodynamic stability within cells is unclear.</p><p>We submit that experiments aimed at measuring the effects of crowding on
reversible associations of native probes are not only more relevant from a
biological standpoint than measurements of protein stability, but also simpler to
interpret, due to the serious theoretical difficulties associated with quantitative
analysis of interactions between flexible unfolded macromolecules and rigid
macromolecules. Furthermore, properly designed probe-based measurements of self-
and/or heteroassociation can be more sensitive to crowding effects than measurements
of stability. For example, several measurements, simulations, and theoretical
estimates82–85 have reported only small effects
of crowding on the formation of homo- or hetero-dimers in solutions of comparably
sized crowding molecules (i.e., increases in equilibrium association constants by
factors of between 2 and 5, or −0.5 <
ΔΔG/RT < −1.5). However, binary
hetero-association results in the formation of a single protein-protein contact
interface, whereas the self-assembly of a larger oligomer (e.g., nP⇄Pn where n > 2) results in the formation of
a minimum of n − 1 intersubunit interfaces and possibly
more, as in the case of a tetragonal tetramer with 6 intersubunit interfaces. When
one multiplies the individually small crowding bias toward the formation of a single
interface by the number of interfaces, the enhancement of oligomer formation by
crowding can become very large. It follows that an experiment designed to follow the
concerted formation of a large oligomer would be expected to be much more sensitive
to excluded volume effects than one designed to follow the formation of a homo- or
heterodimer. Large changes in self-association equilibria were indeed observed in
studies of the effect of crowding on the concerted formation of hexamers of
ClpB34, heptamers of
cp1032 and decamers of
bovine pancreatic trypsin inhibitor86.</p><p>Several of the reported studies of probe stability within crowded media
utilized probes that are smaller than the predominant crowding species57, 87–89. Such
experiments should not be expected to be sensitive to excluded volume effects.
Excluded volume theory predicts that the contribution of excluded volume to a
particular reaction equilibrium will only become substantial when the size of the
probe is comparable to or exceeds that of the major crowding species13 and when the probe reaction
(isomerization or association) is accompanied by a substantial reduction of the
ratio of solvent-exposed surface to volume12. These predictions are borne out by the following
observations: (1) The formation of polymers of sickle hemoglobin (HbS) is
facilitated by high concentrations of bovine serum albumin and hemoglobin mutants
that do not coprecipitate with HbS90; (2) the formation of amyloid fibers of alpha synuclein is
strongly promoted by high concentrations of other proteins as well as by nonionic
polymers91; and (3) the
formation of fiber bundles of FtsZ is strongly facilitated not only by the addition
of traditional polymeric crowding agents, but also by the addition of 10 %
(by volume) of BSA, ovomucoid, and Escherichia coli
lysate92.</p><p>In view of the possible variation of the factors known to contribute to
tracer - crowder interactions, it is evident that information obtained from
measurements on a specific probe in a single (micro)environment under a fixed set of
experimental conditions cannot be reliably generalized. When the abundances and
chemical compositions of the interacting molecules and experimental conditions are
significantly different from those studied, such generalization would likely be
erroneous and misleading in the absence of independently obtained evidence to the
contrary.</p><p>Although recognized long ago66,
79, 93, 94, the importance
of weak, nonspecific interactions as modulators of biochemical processes in vivo has
only recently begun to be widely appreciated5, 11, 65, 95.
Although emphasis has been placed upon attractive interactions and the formation of
weakly associated complexes, it is important to keep in mind that some of the most
influential weak, nonspecific interactions that have been identified as significant
modulators of biochemical equilibria and rates are repulsive steric and
electrostatic interactions between macromolecules that do not form complexes.</p><p>We believe that future advances in our understanding of the influence of
macromolecular crowding upon rates and equilibria of macromolecular reactions in
media as complex and heterogeneous as the interior of a living cell will depend less
upon the development of new tools than upon the willingness of researchers to
utilize experimental and theoretical tools already in hand to build up from the
bottom and, perhaps even more importantly, to build down from the top.</p>