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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Archives of Biochemistry and Biophysics 531 (2013) 4–13 Contents lists available at SciVerse ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi Review The stability of 2-state, 3-state and more-state proteins from simple spectroscopic techniques... plus the structure of the equilibrium intermediates at the same time ⇑ Javier Sancho Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza. Pedro Cerbuna 12, 50009 Zaragoza, Spain Biocomputation and Complex Systems Physics Institute (BIFI), Joint Unit BIFI-IQFR, CSIC, Universidad de Zaragoza. Mariano Esquillor, Edificio I+D, 50018 Zaragoza, Spain article info abstract Article history: Protein stability is not just an academic matter. Biotechnology, Cell Biology and Drug Design are some of Available online 8 November 2012 the fields where both theoretical and practical knowledge of protein stability is required. Simple equip- ment and chemicals, such as a thermostated fluorimeter and common denaturants, suffice to determine the conformational stability of a protein. To this end, the most important experiments are the preliminary ones done to establish the minimum number of species (conformations) accumulating in the equilibrium. For proteins with non-functional equilibrium intermediates, determining the relevant stability of the pro- tein (the free energy difference between the native conformation and the intermediate) is most important, and it allows very valuable structural information on the intermediate to be derived when protein variants are compared to wild type using equilibrium /-analysis. The principles, tricks and equations involved in the analysis of denaturant induced or temperature induced equilibrium unfolding curves by the linear extrapolation method or using the integrated Gibbs–Helmholtz equation, respectively, will be discussed, and a brief outline of challenges and frontiers in the protein stability field will be presented. Ó 2012 Elsevier Inc. All rights reserved. The different types of protein stability a low chemical or biochemical stability is that the population of ac- tive protein molecules will be progressively reduced in an irrevers- Proteins are the molecules that do most things in living beings, ible manner because the reactions involved tend to be irreversible and the wonders they do are almost always related to their molec- and tend to compromise function. Increasing their chemical stabil- ular surfaces and intrinsic dynamics. These, in turn, are shaped by ity is an issue for proteins that are used in biotechnological pro- folding reactions that lead, for each protein, to stabilization of one cesses, which sometimes require extreme conditions such as high specific tridimensional structure among the inconceivably large temperature, low pH, presence of cosolvents, etc. [2]. In contrast, number of possible alternative conformations. In apparent con- the biochemical stability of any given protein, whether high or trast, there are proteins that display unfolded conformations under low, is in principle a functional property that might be thought of native solution conditions [1]. Yet, some of these proteins have as being best left unmodified. However, the increasing availability been observed to become folded when they bind to appropriate of biologics as therapeutic agents [3] makes the rational tailoring molecular targets, which indicates the stabilization of those pro- of biological stability an important goal. teins into defined structures is coupled to binding events. Being The conformational stability of a protein is something different. conformationally stable at some time seems to be a general requi- A protein molecule that leaves the ribosome and becomes folded site of functional proteins. can still experience the inverse process of becoming unfolded It should be noticed that ‘‘protein stability’’ is an ambiguous term and then folded again for as long as its chemical integrity is main- that may refer to several different things (Fig. 1). The chemical or tained. Therefore, for any population of identical protein molecules biochemical stability of a protein is related to its resistance to expe- there is an equilibrium constant (Kf) that governs the fraction of rience changes in its covalent structure, i.e., have some covalent molecules that are folded (vf) or unfolded (vu). bonds cleaved or some atoms replaced. The consequence of having Kf ¼ vf =vu ð1Þ Such an equilibrium constant can be determined using many ⇑ Address: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza. Pedro Cerbuna 12, 50009 Zaragoza, Spain. different techniques and it can be mathematically transformed into Fax: +34 976762123. a free energy difference of folding (DGf) which is usually referred to E-mail address: [email protected] as the conformational stability of the protein. 0003-9861/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2012.10.014 Author's personal copy J. Sancho / Archives of Biochemistry and Biophysics 531 (2013) 4–13 5 DGf ¼RT ln Kf ð2Þ etc. Mechanical stability refers to the resistance of native proteins to be unfolded by external forces, and it is studied with atomic It is easy to get confused with the sign of this magnitude be- force microscopes or optical tweezers [8]. Mechanical unfolding cause there is a tendency in the literature to state that if the free is usually reversible and the magnitude of the force needed to un- energy of folding of a protein is, say, À10 kcal/mol, then its confor- fold a particular protein changes with the orientation of the force. mational stability is 10 kcal/mol. This is in part explained by the This indicates that different transition states may be sampled fact that what is determined and reported in most cases is the free when the force is applied at different points. Thus the mechanical energy of unfolding, which for a stable protein bears, of course, a stability of a protein will be related to the specific energy barrier positive sign. that has to be crossed, given the force applied, to convert native Equilibrium constants are ratios of kinetic constants, and two molecules into unfolded ones. It seems that b proteins tend to dis- proteins with the same value for their folding equilibrium constants play higher mechanical stabilities than a proteins. may differ much in their folding and unfolding rate constants. This It is common that in the folding/unfolding equilibrium of small is not without practical consequences because the reactivity of proteins the only conformations that populate to measurable molar folded and unfolded states [4] may not be the same. A protein rap- fractions are the native, folded state and the unfolded state (U M F) idly sampling the folded and unfolded states may often get a chance [9,10]. In contrast, for most proteins, additional intermediate con- to engage in reactions only possible for or just much faster in the formations (less folded than the native state but more folded than unfolded conformation. If these are irreversible reactions leading the denatured state) accumulate as the solution conditions become to loss of function, a much greater fraction of such a protein may be- more destabilizing of the native conformation, yet not fully stabiliz- come inactive in a given time period than in a protein with the same ing of the denatured state [11]. The number of species that will ap- conformational stability but with slow unfolding kinetics. Proteins pear as the solution conditions are shifted from native to denaturing whose native conformation is preserved from experiencing inacti- will depend on the specific shape of the folding landscape in native vation related to reactions of its unfolded conformation due to their conditions and on the changes that the specific denaturing agent slow unfolding kinetics are considered to display a high kinetic sta- used to probe the equilibrium will induce in that shape. The more bility [5]. It seems now that aggregates rich in b-strands may be for complex the landscape, the more likely a higher number of interme- many, perhaps most, proteins more stable than their corresponding diates will be observed. On the other hand, a particular denaturing native conformations [6]. It is thus possible that proteins have agent (say, urea) may not reveal the presence of a potential interme- evolved to protect their native conformations from transformation diate while other (say, temperature) may do so [12]. For proteins into more stable aggregated conformations such as those mani- displaying equilibrium intermediates, the global equilibrium be- fested in aggregative diseases [7]. A high energy barrier between tween the native and the unfolded states can be divided