Simulating Crowding In Vitro: Not an Elusive Goal Any Longer.

A few decades ago, it became clear that most biophysical studies were performed on proteins under conditions not corresponding to those found in vivo, thus limiting the generality of at least some of the conclusions. The main difference was identified in the absence, in the dilute solutions typically used, of large amounts of other macromolecules. Solution conditions similar to those found in vivo were dubbed as crowded. The main consequences of crowding were identified as the influence of crowding on protein stability and on alterations of reaction conditions due to changes in protein activity. Initial experimental work to assess the effect of crowding on protein stability was dominated by theoretical estimations that suggested an increase of the unfolding temperatures between 5 and 20 °C. The present chapter examines some of the techniques typically used in these studies in a critical way, exploring the most common methodologies employed to study proteins under crowded conditions. One of the critical problems is the choice of the crowders. There are two categories of commonly used macromolecular crowders, synthetic polymers and common proteins. Synthetic crowders often do not interfere with optical measurements of proteins. When proteins are used as crowder it is necessary to label the protein under study. We demonstrate that studies of protein stability in crowded solutions provide more valuable information when observing the behaviour of marginally stable proteins.