Review
Crowding revisited: Open questions and future perspectives

https://doi.org/10.1016/j.tibs.2022.05.007Get rights and content

Highlights

  • The interior of cells is an aqueous solution much more concentrated than buffers used for in vitro studies, posing the problem of how such ‘crowded’ conditions influence protein stability and other properties.

  • The presence of high concentrations of crowders limits the volume available to proteins under study and impacts their thermal stability.

  • The influence of crowders on protein stability is modulated by the presence of enthalpic contributions, which may shield the real mechanism of crowding.

  • Careful studies that minimise weak interactions support the importance of entropic contributions.

Although biophysical studies have traditionally been performed in diluted solutions, it was pointed out in the late 1990s that the cellular milieu contains several other macromolecules, creating a condition of molecular crowding. How crowding affects protein stability is an important question heatedly discussed over the past 20 years. Theoretical estimations have suggested a 5–20°C effect of fold stabilisation. This estimate, however, is at variance with what has been verified experimentally that proposes only a limited increase of stability, opening the question whether some of the assumptions taken for granted should be reconsidered. The present review critically analyses the causes of this discrepancy and discusses the limitations and implications of the current concept of crowding.

Section snippets

The birth of the crowding concept

Most biophysical and biochemical studies on proteins are performed in dilute buffer solutions. It has been noted, however, that the cell interior contains high amounts of macromolecules and thus ought to be described as ‘crowded.’ The concept of crowding (see Glossary) in cells was introduced by Alexander George Ogston [1,2] but became popular only after the first articles of Allen Minton [3., 4., 5.]. To understand how crowding plays a role in protein behaviour, it was first assumed that

How much does crowding influence protein stability?

The influence of macromolecular cosolutes, although not explicitly referring to the comparison with cellular conditions, is not a new concept. It was described by Ogston [2] and, many years earlier, by Asakura and Oosawa [22,23], and again more recently by Oosawa [24]. Nevertheless, from 1980, when Minton first proposed the concept of crowding, to 2000, when he explicitly published an elegant, simple model to account for the importance of excluded volume for protein stability [5], there was a

Thermal resistance or thermal stability?

When people talk about ‘protein stability,’ they often use the term loosely and might use a number of different quantities (Tm, ΔH, etc.) to quantify the concept. These can be equivalent (or proportional) but do not need to be so, and one has to be very careful. The temperature dependence of a parameter during heat unfolding detected with different techniques (Box 1), such as the intensity of a circular dichroism (CD) spectrum at 222 nm, can be used to derive two thermodynamic parameters of the

The importance of estimating protein volume correctly

Another important aspect of understanding the discrepancy between expected and experimental stability is how protein volume measurements are interpreted. At the origin of studies on the influence of crowding on protein stability, there are estimates of volume changes of proteins upon unfolding [5,31]. The volume of the folded species can often be accurately measured by experimental studies, including techniques such as X-ray crystallography. Many of the folded species have a shape resembling an

Entropy or enthalpy? This is the question…

Important theoretical contributions to the concept of crowding have also been made by Sapir and Harries [41,42], who went back to the origin of protein stabilisation as it emerges in crowded solutions. Not many people in the field of crowding know that the first mention of this concept dates to the so-called depletion theory or Asakura-Oosawa model, put forward many years before Minton or even Ogston [22,23]. According to the Asakura-Oosawa model, when there is a sizeable attraction between

Molecular crowding or cellular environment mimicking?

The extensive work by the Pielak lab [10., 11., 12., 13., 14., 15., 16., 17., 18., 19., 20.] and others has been essential to understand the ‘real’ environment inside cells. However, the contraposition of entropy versus enthalpy has not directly clarified the contribution of crowding on protein stability. It has been known for a long time that chemical interactions between proteins and between proteins and small molecules are of paramount importance, and it is nearly impossible to study systems

Simulations and similarities

In addition to experimental approaches, computational methods have helped in understanding the role of crowding on protein stability. Several simulation studies have, for instance, extensively investigated how cellular crowders may influence the folding and function of protein domains [33,47., 48., 49., 50.]. It was concluded that molecular crowders can both stabilise and destabilise proteins, depending on the nature of both the crowder and the protein. A much cited such example is the

Concluding remarks

It is fair to say that many measurements of protein stability in crowded environments are not always fully reliable, because it ought to be desirable to measure the whole stability curve of the protein before drawing final assessments on the influence of crowding. The concept of macromolecular crowding was thoughtfully introduced by Allen Minton in the 1980s to draw attention to a macroscopic difference between studies in vitro, usually performed on very dilute buffered solutions, and

Declaration of interests

The authors have no interests to declare.

Glossary

Biosensors
devices that generate a signal proportional to the concentration of a compound, incorporating a biological sensing element.
Brownian walk
strictly speaking, in mathematics, this term describes a path made of a succession of random steps; in this review, it is used to describe the denatured state as a random coil without internal excluded volume.
Circular dichroism
a spectroscopic technique used to analyse chirality in molecules; in the case of proteins, made by chiral amino acid residues,

References (67)

  • T.C. Laurent et al.

    The interaction between polysaccharides and other macromolecules. 4. The osmotic pressure of mixtures of serum albumin and hyaluronic acid

    Biochem. J.

    (1963)
  • A.G. Ogston

    On the interaction of solute molecules with porous networks

    J. Phys. Chem.

    (1970)
  • A.P. Minton

    The effect of volume occupancy upon the thermodynamic activity of proteins: Some biochemical consequences

    Mol. Cell. Biochem.

    (1983)
  • J.S. Clegg

    Properties and metabolism of the aqueous cytoplasm and its boundaries

    Am. J. Phys.

    (1984)
  • F.X. Theillet

    Physicochemical properties of cells and their effects on intrinsically disordered proteins (IDPs)

    Chem. Rev.

    (2014)
  • A.C. Miklos

    Volume exclusion and soft interaction effects on protein stability under crowded conditions

    Biochemistry

    (2010)
  • A.C. Miklos

    Protein crowding tunes protein stability

    J. Am. Chem. Soc.

    (2011)
  • Y. Wang

    Macromolecular crowding and protein stability

    J. Am. Chem. Soc.

    (2012)
  • M. Sarkar

    Soft interactions and crowding

    Biophys. Rev.

    (2013)
  • M. Senske

    Protein stabilization by macromolecular crowding through enthalpy rather than entropy

    J. Am. Chem. Soc.

    (2014)
  • R.D. Cohen

    Intracellular pH modulates quinary structure

    Protein Sci.

    (2015)
  • R.D. Cohen et al.

    Electrostatic contributions to protein quinary structure

    J. Am. Chem. Soc.

    (2016)
  • R.D. Cohen et al.

    A cell is more than the sum of its (dilute) parts: A brief history of quinary structure

    Protein Sci.

    (2017)
  • P.R. Davis-Searles

    Interpreting the effects of small uncharged solutes on protein-folding equilibria

    Annu. Rev. Biophys. Biomol. Struct.

    (2001)
  • W.B. Monteith

    Quinary structure modulates protein stability in cells

    Proc. Natl. Acad. Sci. U. S. A.

    (2015)
  • K.A. Sharp

    Analysis of the size dependence of macromolecular crowding shows that smaller is better

    Proc. Natl. Acad. Sci. U. S. A.

    (2015)
  • S. Asakura et al.

    Interaction between particles suspended in solutions of macromolecules

    J. Polym. Sci.

    (1958)
  • S. Asakura et al.

    On interaction between two bodies immersed in solution of macromolecules

    J. Chem. Phys.

    (1954)
  • F. Oosawa

    The history of the birth of the Asakura-Oosawa theory

    J. Chem. Phys.

    (2021)
  • B. Köhn et al.

    Macromolecular crowding tunes protein stability by manipulating solvent accessibility

    Chembiochem

    (2019)
  • S. Denos

    Crowding effects on the small, fast-folding protein lambda6-85

    Faraday Discuss.

    (2012)
  • Y. Wang

    Effect of Ficoll 70 on thermal stability and structure of creatine kinase

    Biochem. Mosc.

    (2010)
  • X. Aguilar

    Macromolecular crowding extended to a heptameric system: The co-chaperonin protein 10

    Biochemistry

    (2011)
  • Cited by (11)

    View all citing articles on Scopus
    2

    Present address: European Synchrotron Radiation Facility, Grenoble, France

    View full text