Trends in Biochemical Sciences
ReviewCrowding revisited: Open questions and future perspectives
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,
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Present address: European Synchrotron Radiation Facility, Grenoble, France