Double-mutant cycles: a powerful tool for analyzing protein structure and function

A double-mutant cycle involves wild-type protein, two single mutants and the corresponding double mutant protein. If the change in free energy associated with a structural or functional property of the protein upon a double mutation differs from the sum of changes in free energy due to the single mutations, then the residues at the two positions are coupled. Such coupling reflects either direct or indirect interactions between these residues. Double-mutant cycle analysis can be used to measure the strength of intramolecular and intermolecular pairwise interactions in proteins or protein–ligand complexes with known structure. Double-mutant cycles can also be employed to characterize structures that are inaccessible to NMR and X-ray crystallography, such as those of transition states for protein folding, ligand binding and enzyme catalysis, or of membrane proteins. Multidimensional mutant cycle analysis can be used to measure higher-order cooperativity between intramolecular or intermolecular interactions. In the absence of coupling between residues, prediction of mutational effects is possible by assuming their additivity

A Method for Removing Effects of Nonspecific Binding on the Distribution of Binding Stoichiometries: Application to Mass Spectroscopy Data

There is often an interest in knowing, for a given ligand concentration, how many protein molecules have one, two, three, etc. ligands bound in a specific manner. This is a question that cannot be addressed using conventional ensemble techniques. Here, a mathematical method is presented for separating specific from nonspecific binding in nonensemble studies. The method provides a way to determine the distribution of specific binding stoichiometries at any ligand concentration when using nonensemble (e.g., single-molecule) methods. The applicability of the method is demonstrated for ADP binding to creatine kinase using mass spectroscopy data. A major advantage of our method, which can be applied to any protein-ligand system, is that no previous information regarding the mechanism of ligand interaction is required

N-Terminal Domains in Two-Domain Proteins Are Biased to Be Shorter and Predicted to Fold Faster Than Their C-Terminal Counterparts

SummaryComputational analysis of proteomes in all kingdoms of life reveals a strong tendency for N-terminal domains in two-domain proteins to have shorter sequences than their neighboring C-terminal domains. Given that folding rates are affected by chain length, we asked whether the tendency for N-terminal domains to be shorter than their neighboring C-terminal domains reflects selection for faster-folding N-terminal domains. Calculations of absolute contact order, another predictor of folding rate, provide additional evidence that N-terminal domains tend to fold faster than their neighboring C-terminal domains. A possible explanation for this bias, which is more pronounced in prokaryotes than in eukaryotes, is that faster folding of N-terminal domains reduces the risk for protein aggregation during folding by preventing formation of nonnative interdomain interactions. This explanation is supported by our finding that two-domain proteins with a shorter N-terminal domain are much more abundant than those with a shorter C-terminal domain

Analysing the origin of long-range interactions in proteins using lattice models

<p>Abstract</p> <p>Background</p> <p>Long-range communication is very common in proteins but the physical basis of this phenomenon remains unclear. In order to gain insight into this problem, we decided to explore whether long-range interactions exist in lattice models of proteins. Lattice models of proteins have proven to capture some of the basic properties of real proteins and, thus, can be used for elucidating general principles of protein stability and folding.</p> <p>Results</p> <p>Using a computational version of double-mutant cycle analysis, we show that long-range interactions emerge in lattice models even though they are not an input feature of them. The coupling energy of both short- and long-range pairwise interactions is found to become more positive (destabilizing) in a linear fashion with increasing 'contact-frequency', an entropic term that corresponds to the fraction of states in the conformational ensemble of the sequence in which the pair of residues is in contact. A mathematical derivation of the linear dependence of the coupling energy on 'contact-frequency' is provided.</p> <p>Conclusion</p> <p>Our work shows how 'contact-frequency' should be taken into account in attempts to stabilize proteins by introducing (or stabilizing) contacts in the native state and/or through 'negative design' of non-native contacts.</p

Trade-off between Positive and Negative Design of Protein Stability: From Lattice Models to Real Proteins

Two different strategies for stabilizing proteins are (i) positive design in which the native state is stabilized and (ii) negative design in which competing non-native conformations are destabilized. Here, the circumstances under which one strategy might be favored over the other are explored in the case of lattice models of proteins and then generalized and discussed with regard to real proteins. The balance between positive and negative design of proteins is found to be determined by their average ‘‘contact-frequency’’, a property that corresponds to the fraction of states in the conformational ensemble of the sequence in which a pair of residues is in contact. Lattice model proteins with a high average contact-frequency are found to use negative design more than model proteins with a low average contact-frequency. A mathematical derivation of this result indicates that it is general and likely to hold also for real proteins. Comparison of the results of correlated mutation analysis for real proteins with typical contact-frequencies to those of proteins likely to have high contactfrequencies (such as disordered proteins and proteins that are dependent on chaperonins for their folding) indicates that the latter tend to have stronger interactions between residues that are not in contact in their native conformation. Hence, our work indicates that negative design is employed when insufficient stabilization is achieved via positive design owing to high contact-frequencies