Liaison amid disorder: non-native interactions may underpin long-range coupling in proteins
© BioMed Central Ltd 2009
Published: 13 March 2009
A lattice-model study of double-mutant cycles published in BMC Structural Biology underscores how interactions in non-native conformations can lead to thermodynamic coupling between distant residues in globular proteins, adding to recent advances in delineating the often crucial roles played by disordered conformational ensembles in protein behavior.
How do the conformational structures, dynamics and biological function of a protein emerge from the interactions among its amino acid residues? A significant part of current ideas about protein behaviors is based on structures in the Protein Data Bank (PDB) and notions of contact-like interactions between amino acid residues in spatial proximity. While useful, this picture is limited. In particular, studies of allostery and mutational analyses have demonstrated that energetic coupling can exist between residues at positions far apart in a protein's native structure. An intriguing possibility is that such apparently long-range coupling may arise from the residues' transient association in the unfolded state. This scenario was elucidated by an extensive computational study using two-dimensional lattice protein models published recently in BMC Structural Biology by the groups of Ron Unger and Amnon Horovitz (Noivirt-Brik et al. ). Their study provides a theoretical framework that will be useful for guiding future experiments. It also highlights the power and versatility of simple lattice modeling. Despite the highly coarse-grained representations of polypeptide chains used, this decades-old practice offers conceptual clarity and has been proved effective time and again in discovering and elucidating fundamental biophysical principles.
Characterizing energetic coupling by double-mutant cycle
Energetic coupling between amino acid residues is difficult to discern from the static folded structure of a protein alone. Double-mutant cycle (DMC) is a direct perturbative technique to assess the degree to which the consequences of mutations at two different sites are correlated. DMC compares the sum of effects of two single mutations on two sites (one at a time) and the effect of double mutations on both of the sites. Often, as in Noivirt-Brik et al. , the effect of interest is the free energy of folding, ΔG (native state more stable for more negative ΔG). If ΔΔG(m1), ΔΔG(m2), and ΔΔG(m1, m2) are, respectively, the changes in ΔG resulting from two single mutations and from the double mutations (ΔΔG equals ΔG of the mutant minus that of the wild type), coupling is quantified by an 'interaction free energy' ΔΔGint = ΔΔG(m1, m2) – [ΔΔG(m1) + ΔΔG(m2)]. The two sites are energetically independent if the mutational effects are additive (ΔΔGint = 0). Otherwise they are coupled, wherein the native state is either stabilized (ΔΔGint < 0) or destabilized (ΔΔGint > 0) by coupling. Energetic coupling may also be estimated using a bioinformatics approach based on evolutionary assumptions. This indirect method has also identified likely long-range interactions, for example in PDZ domains .
Long-range coupling in proteins can have multiple physical origins
Reverse hydrophobic effect and other manifestations of non-native interactions
Does the model in Noivirt-Brik et al.  and that shown in Figure 1b reasonably mimic reality? Ample evidence supports the existence of non-native interactions in protein unfolded states . As early as 1990, the hydrophobicity of an exposed residue in the Cro repressor from bacteriophage λ was found to correlate negatively with the stability of the protein. Dubbed the 'reverse hydrophobic effect' to contrast it with the usual role of hydrophobicity in stabilizing the folded state, the phenomenon was rationalized by the proposal that the residue is partially buried; that is, it has non-native contact(s) in the unfolded state . The variation in the denaturant dependence of native stability (equilibrium m-value, defined as the rate of decrease in native stability with respect to increase in denaturant concentration) of staphylococcal nuclease observed in earlier site-directed mutagenesis experiments also indicated variable hydrophobic burial in the unfolded state. Recent experiments suggested that non-native ionic interactions are present as well in the unfolded states of the amino-terminal domain of ribosomal protein L9 (see  and references therein).
Simple lattice protein models are an effective conceptual tool
Lattice models have been successful in accounting for some of these phenomena. An early HP square-lattice model study elucidated how mutations can lead to substantial changes in m-value, as found for staphylococcal nuclease experimentally . Figure 1b shows three HP model mutants that exhibit reverse hydrophobic effect (ΔΔG < 0). From their ΔΔG values, ΔΔGint for the model DMC was determined to be significantly negative (green curve in the left plot of Figure 1b). This result indicates a long-range coupling (between the red and blue residues) underpinned by non-native interactions in the unfolded state of the HP model.
As illustrated by these examples and similar analyses by Noivirt-Brik et al. , lattice models are a powerful investigative tool. Common notions about protein energetics are sometimes fuzzy. Their precise ramifications are often obscure owing to a lack of discipline from an explicit consideration of chain connectivity and conformational entropy . Lattice models account for these key ingredients, albeit in a simplified fashion. By virtue of their computational tractability, lattice models can clarify the logic between assumptions and testable consequences, generate new hypotheses, and ask 'what if' questions to advance conceptual understanding.
It goes without saying that lattice models are limited. Learning from both their strengths and limitations, concrete progress often requires comparative evaluation of models embodying different physical ideas. Notably, extensive analyses over the past decade have shown that traditional lattice protein models – the HP model included – fold much less cooperatively than real, two-state proteins . In the light of this knowledge, it is instructive to explore whether the predictions about long-range coupling obtained by Noivirt-Brik et al.  and from the HP model are robust.
Folding cooperativity may dampen but cannot eliminate non-native interactions
Contact interactions such as that in the model used by Noivirt-Brik et al.  and the HP model do not fully capture protein energetics. More subtle physical chemistry has enabled higher native specificity and more cooperative folding to be achieved in natural proteins. Hence, the probabilities of non-native interactions and the long-range coupling they engender in real proteins are likely to be lower than those stipulated by these models. This point is illustrated in Figure 1b using a class of energy functions E = (1 - s) EHP + sEGō interpolating between the HP model (EHP) and a Gō model (EGō) that favors only native interactions (formulated originally by Nobuhiro Gō and co-workers in 1975; see reference to Gō in ). Here, s is the weight of Gō energy and thus a parameter for native specificity. As the strength of favorable non-native interactions decreases with increasing s, the associated long-range coupling diminishes. Could all non-native interactions be 'designed out' by evolution?
From biophysics to biological functions of non-native protein conformations
The main point of the study of Noivirt-Brik et al.  – that non-native interactions are the origin of some long-range coupling – is thus on a firm physical and molecular biological footing. A deeper question is whether non-native interactions are mere annoying necessities imposed by physics, a feature that should be designed out if possible by evolution, or whether they can serve biological purposes? With our increasing appreciation of the regulatory functions of intrinsically disordered proteins , there is no reason to believe that biology would not exploit every opportunity presented by physics. A case in point is that non-native conformations can have 'promiscuous' biological functions different from the dominant function of a protein, and that selection for promiscuous functions can speed up evolution considerably . In this case as well, simple lattice modeling has afforded the pertinent biophysical principles (see accompanying article of ). More discoveries lie ahead as protein scientists broaden our sight beyond well-ordered folded native structures.
We acknowledge support from the Canada Research Chairs Program and funding from the Canadian Institutes of Health Research to HSC (grant MOP-84281). We thank Matt Cordes of University of Arizona for a helpful discussion on the λ Cro repressor. Because journal policy placed a limit on the number of references, we apologize to colleagues whose important contributions we were not able to cite.
- Noivirt-Brik O, Unger R, Horovitz A: Analysing the origin of long-range interactions in proteins using lattice models. BMC Struct Biol. 2009, 9: 4-PubMedPubMed CentralView Article
- Lockless SW, Ranganathan R: Evolutionarily conserved pathways of energetic connectivity in protein families. Science. 1999, 286: 295-299.PubMedView Article
- Haliloglu T, Bahar I, Erman B: Gaussian dynamics of folded proteins. Phys Rev Lett. 1997, 79: 3090-3093.View Article
- Shortle D, Chan HS, Dill KA: Modeling the effects of mutations on the denatured states of proteins. Protein Sci. 1992, 1: 201-215.PubMedPubMed CentralView Article
- Bowler BE: Thermodynamics of protein denatured states. Mol BioSyst. 2007, 3: 88-99.PubMedView Article
- Pakula AA, Sauer RT: Reverse hydrophobic effects relieved by amino-acid substitutions at a protein surface. Nature. 1990, 344: 363-364.PubMedView Article
- Chan HS, Shimizu S, Kaya H: Cooperativity principles in protein folding. Methods Enzymol. 2004, 380: 350-379.PubMedView Article
- Muñoz V, Lopez EM, Jager M, Serrano L: Kinetic characterization of the chemotactic protein from Escherichia coli, CheY. Kinetic analysis of the inverse hydrophobic effect. Biochemistry. 1994, 33: 5858-5866.PubMedView Article
- Cranz-Mileva S, Friel CT, Radford SE: Helix stability and hydrophobicity in the folding mechanism of the bacterial immunity protein Im9. Protein Eng Des Sel. 2005, 18: 41-50.PubMedView Article
- Zarrine-Afsar A, Wallin S, Neculai AM, Neudecker P, Howell PL, Davidson AR, Chan HS: Theoretical and experimental demonstration of the importance of specific nonnative interactions in protein folding. Proc Natl Acad Sci USA. 2008, 105: 9999-10004.PubMedPubMed CentralView Article
- Mittag T, Forman-Kay JD: Atomic-level characterization of disordered protein ensembles. Curr Opin Struct Biol. 2007, 17: 3-14.PubMedView Article
- Amitai G, Gupta RD, Tawfik DS: Latent evolutionary potentials under the neutral mutational drift of an enzyme. HFSP J. 2007, 1: 67-78.PubMedPubMed CentralView Article