Downloaded from http://cshperspectives.cshlp.org/ on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Protein Solubility Predictions Using the CamSol Method in the Study of Protein Homeostasis Pietro Sormanni and Michele Vendruscolo Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom Correspondence: [email protected] One of the major functions of the protein homeostasis system is to maintain proteins in their soluble states, and indeed several human disorders are associated with the aberrant aggrega- tion of proteins. An active involvement of the protein homeostasis system is necessary to avoid aggregation because proteins are expressed at levels close to their solubility limits, hence being poorly soluble. The mechanisms by which the protein homeostasis system acts to control protein aggregation are, however, still not known in much detail. To facilitate system- atic investigations of these mechanisms, we describe here the CamSol method of predicting protein solubility, and illustrate its initial applications. We anticipate that with the advent of powerful proteomics and transcriptomic methods, in combination with the use of the CamSol method and related approaches to predict the solubility and other biophysical properties of proteins, it will become possible to increase our understanding of the principles of protein homeostasis related to the maintenance of the proteome in its soluble form. PROTEIN SOLUBILITY One can also think about the solubility as the particular concentration for which adding more he solubility of a substance is defined by the substance does not increase its concentration in Tvalue of the concentration at which soluble solution, but triggers instead its precipitation. and insoluble phases are in equilibrium (Chai- As a wide variety of proteins are functional kin et al. 1995). The solubility therefore funda- in their soluble forms, and are associated with mentally depends on the physical and chemical disease in their insoluble forms, protein solubil- properties of the substance itself, as well as on ity is a major aspect of protein homeostasis the properties of its environment, including in (Vendruscolo et al. 2011). Defining the solubil- particular the composition of the solvent, the ity of proteins, however, is difficult both theoret- temperature, the pH, and the ionic strength. Op- ically and experimentally (Jain et al. 2017; Wolf eratively, the solubility of a substance can be Pérez et al. 2019). As given above, the definition measured by its saturation concentration, also of solubility is rigorous, but it only applies in a known as critical concentration, which is the fully quantitative manner to substances that concentration of the soluble fraction of the sub- have well-defined soluble and insoluble phases. stance in the presence of an insoluble fraction. The vast majority of proteins, however, can pop- Editors: Richard I. Morimoto, F. Ulrich Hartl, and Jeffery W. Kelly Additional Perspectives on Protein Homeostasis available at www.cshperspectives.org Copyright © 2019 Cold Spring Harbor Laboratory Press; all rights reserved Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a033845 1 Downloaded from http://cshperspectives.cshlp.org/ on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press P. Sormanni and M. Vendruscolo ulate a wide range of structurally heterogeneous these conditions may be more or less harsh in states, including small and large oligomers, and terms of pH, concentration, temperature, or it is thus challenging to distinguish between presence of chemicals (e.g., trifluoroethanol, soluble and insoluble assemblies (Sormanni TFE), but essentially all proteins can aggregate et al. 2015). In principle, oligomers capable of (Dobson 1999; Knowles et al. 2014). growing into larger aggregates can be considered The fundamental nature of the aggregated as insoluble, but it is, in practice, difficult to state and its manifestations in living organisms measure the concentrations of growth-compe- has been increasingly recognized and has led to tent oligomers in a given sample. Despite this the observation of the phenomenon of wide- problem, it is still useful to consider protein sol- spread protein aggregation, whereby a large frac- ubility measurements in the study of protein tion of the proteome undergoes aggregation in homeostasis because the overall concentration vivo (David et al. 2010; Olzscha et al. 2011; Reis- of the oligomeric species is typically much Rodrigues et al. 2012; Ciryam et al. 2013; Wal- smaller than that of monomers and large aggre- ther et al. 2015). It has been realized that the gates, so that the contribution of these oligomer- origin of this phenomenon is that proteins are ic species to the value of the solubility is relative- expressed at levels close to their solubility limits ly small (Sormanni et al. 2015). (Tartaglia et al. 2007; Baldwin et al. 2011), there- fore being supersaturated (Ciryam et al. 2015). Taken together, all these observations indicate PROTEIN SOLUBILITY, PROTEIN that the ability to aggregate and form fibrils can HOMEOSTASIS, AND HUMAN DISEASE be regarded as an intrinsic property of polypep- The phenomenon of protein aggregation is as- tide chains, even if the aggregation rates can vary sociated with a wide range of human disorders, dramatically among different proteins. To re- including Alzheimer’s and Parkinson’s diseases spond to the constant risk of aggregation, a so- (Dobson 1999; Balch et al. 2008; Eisenberg and phisticated protein homeostasis system has Jucker 2012; Knowles et al. 2014). These diseases evolved to maintain the proteome in a function- can have different symptoms and affect different al state (Balch et al. 2008; Hartl et al. 2011; organs and tissues, but are all characterized by Labbadia and Morimoto 2015). the dysfunctional aggregation of specificpro- Folded proteins can aggregate through at teins. Although these diseases are associated least two possible paths, depending on the pro- with proteins with different sequences and na- tein under scrutiny and the experimental con- tive structures, most of these proteins seem to ditions (Chiti and Dobson 2017). In one path, follow a generic behavior in misfolding and ag- protein molecules unfold at least partially before gregation (Dobson 1999; Knowles et al. 2014). associating with each other. This process results Such behavior involves the formation of many in the formation of small oligomers, which can different types of aggregates, from small oligo- then grow in size forming more structured ag- mers to large amyloid fibrils, yet all these cate- gregates. Examples of proteins in this class are gories of aggregates appear to share common serpins, where aggregation is believed to initiate structural features that do not depend on the from a folding intermediate (Carrell and Lomas particular proteins they originated from (Dob- 1997). In an alternative path, proteins may ag- son 1999; Balch et al. 2008; Eisenberg and Jucker gregate directly from their native structures 2012; Knowles et al. 2014). Indeed, it is now well- forming a first assembly that can remain func- established that almost every protein, and not tional. This is the case, for instance, for most just those that are disease-related, can aggregate antibody molecules, and other proteins that ex- in vitro under appropriate experimental con- pose “sticky” regions for functional reasons, to ditions, and the resulting aggregates can be toxic bind other proteins or to form complexes (Pech- for cells (Bucciantini et al. 2002; Selkoe 2003; mann et al. 2009). Once a critical number of Eisenberg and Jucker 2012; Chiti and Dobson molecules is present, so that the enthalpy asso- 2017). Depending on the protein under scrutiny, ciated with their ordered stacking overcomes the 2 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a033845 Downloaded from http://cshperspectives.cshlp.org/ on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Protein Solubility Predictions Using CamSol corresponding loss of entropy, the aggregates der native conditions, have more chances to ex- may evolve into protofibrils and ultimately pose hydrophobic residues that can promote ag- into amyloid fibrils (Knowles et al. 2014). In gregation (Tartaglia et al. 2008; Tartaglia and this context we note that increasing amounts Vendruscolo 2008). These principles have been of evidence suggest that the small oligomers, used for the development of a wide range of rather than the large aggregates or amyloid fi- methods for predicting the aggregation propen- brils, may be the most neurotoxic species (Lam- sity (Fernandez-Escamilla et al. 2004; Tartaglia bert et al. 1998; Bucciantini et al. 2002; Haass and Vendruscolo 2008; Maurer-Stroh et al. 2010; and Selkoe 2007; Benilova et al. 2012; Jongbloed Zambrano et al. 2015; Pallarès and Ventura et al. 2015; Cline et al. 2018). 2016) and solubility (Magnan et al. 2009; Agos- tini et al. 2012; Smialowski et al. 2012) of pro- teins. Here we describe in particular the work BIOPHYSICAL PRINCIPLES FOR THE that has led to the introduction of the CamSol PREDICTION OF PROTEIN AGGREGATION method (Sormanni et al. 2015) and illustrate PROPENSITY some of its applications. Although amyloid fibrils and amorphous aggre- gates are fundamental states of polypeptide chains (Knowles et al. 2014), the propensity to THE CamSOL METHOD aggregate