Kenneth P. Murphy

Evaluation of linked protonation effects in protein binding reactions using isothermal titration calorimetry

A theoretical development in the evaluation of proton linkage in protein binding reactions by isothermal titration calorimetry (ITC) is presented. For a system in which binding is linked to protonation of an ionizable group on a protein, we show that by performing experiments as a function of pH in buffers with varying ionization enthalpy, one can determine the pK(a)s of the group responsible for the proton linkage in the free and the liganded states, the protonation enthalpy for this group in these states, as well as the intrinsic energetics for ligand binding (delta H(o), delta S(o), and delta C(p)). Determination of intrinsic energetics in this fashion allows for comparison with energetics calculated empirically from structural information. It is shown that in addition to variation of the ligand binding constant with pH, the observed binding enthalpy and heat capacity change can undergo extreme deviations from their intrinsic values, depending upon pH and buffer conditions.

Urea effects on protein stability: Hydrogen bonding and the hydrophobic effect

The effects of urea on protein stability have been studied using a model system in which we have determined the energetics of dissolution of a homologous series of cyclic dipeptides into aqueous urea solutions of varying concentration at 25°C using calorimetry. The data support a model in which urea denatures proteins by decreasing the hydrophobic effect and by directly binding to the amide units via hydrogen bonds. The data indicate also that the enthalpy of amide hydrogen bond formation in water is considerably higher than previously estimated. Previous estimates included the contribution of hydrophobic transfer of the α‐carbon resulting in an overestimate of the binding between urea and the amide unit of the backbone and an underestimate of the binding enthalpy. Proteins 31:107–115, 1998.

Variability in the pKa of histidine side-chains correlates with burial within proteins

Acidic pKas of histidines buried within the protein interior are frequently rationalized on the contradictory basis of either polar interactions within the protein or the effects of a hydrophobic environment. To examine these relationships, we surveyed the buried surface area, depth of burial, polar interactions, and crystallographic temperature factors of histidines of known pKa. It has been found that buried environments of histidines do not always result in acidic pKas. Instead, the variability of histidine pKas increases for residues where the majority of the side‐chain is buried. Because buried histidines are always found in mixed polar/apolar environments, multiple environmental contributions to pKa values must be considered. However, the quantitative relationships between heterogeneous environments and pKa values are not immediately apparent from the available data. Proteins 2002;49:1–6.

PREDICTION OF BINDING ENERGETICS FROM STRUCTURE USING EMPIRICAL PARAMETERIZATION

We have presented an empirical method that can be used to predict the binding energetics for protein-protein or protein-peptide interactions from three-dimensional structures. The approach differs from other empirical methods in yielding a thermodynamic description of the binding process, including delta Cp, delta H degree, and delta S degree, rather than predicting delta G degree alone. These thermodynamic terms can provide a wealth of detail about the nature of the interaction, and, if sufficient experimental data are available for comparison, a greater assessment of the accuracy of the calculations. A recurring theme throughout this article is the need for more complete thermodynamic and structural characterizations of protein-ligand interactions. This includes not only characterization of the binding delta H degree, delta S degree, and delta Cp, but a thorough investigation into equilibria linked to binding, such as protonation, ion binding, and conformational changes. Sufficient data will allow parameterization on binding data rather than protein unfolding data. Further inclusion of information obtained from unfolding studies is not likely to generate significant improvement in the accuracy of the calculations. As additional binding data become available, the parameterization can be further extended to include relationships derived from analyses of these data. Not only will this increase accuracy and thus confidence, but allow extension of the method of additional types of interactions.

Structural energetics of protein folding and binding

Structural energetics is a method for calculating the energetics of protein folding and binding reactions as a function of temperature. This approach allows measured energetics to be interpreted with regards to the protein structure and the prediction of energetics from known structures. Recent advances include improvements in the parameterization of enthalpy, entropy and heat capacity terms and new applications, especially with regards to understanding dynamic properties of proteins and how these are affected by ligand binding.

Predicting binding energetics from structure: Looking beyond ?G

Structure‐based design of pharmaceuticals requires the ability to predict ligand affinity based on knowledge of structure. The primary term of interest is the binding affinity constant, K, or the free energy of binding, ΔG°. It is common to attempt to predict ΔG° based on empirically derived terms which represent common contributions such as the hydrophobic effect, hydrogen bonding, and conformational entropy. Although these approaches have met with some success, when they fail it is difficult to know which parameter(s) need refinement. Confidence in these approaches is also limited by the fact that ΔG° typically is made up of compensating enthalpic and entropic terms, ΔH° and ΔS°, so that accurate prediction of a ΔG° value may be fortuitous and may not indicate a reasonable understanding of the underlying relationship between structure and affinity. This is further complicated by the fact that both ΔH° and ΔS° are strongly temperature dependent through the heat capacity change, ΔCp. In order to avoid these difficulties, we attempt to use structural data to predict ΔH°, ΔS°, and ΔCp from which ΔG° can be calculated as a function of temperature. The predictions are then compared to experimentally determined values. These calculations have been applied to several systems by ourselves and others. Systems include the binding of angiotensin II to an antibody, the dimerization of interleukin‐8, and the binding of inhibitors to aspartic and serine proteases. Overall the calculations are very successful, and suggest that our understanding of the contributions of the hydrophobic effect, hydrogen bonding, and conformational entropy are quite good. Several of these systems show a strong dependence of the binding energetics on pH, indicative of changes in proton affinity of ionizable groups upon binding. It is critical to account for these protonation contributions to the binding energetics in order to assess the reliability of any computational prediction of energetics from structure. Methods have been developed for determining the energetics of proton binding using isothermal titration calorimetry. The availability of these methods provides a means of understanding how protein structure can modify the pKas of ionizable groups. This information will further add to our understanding of structural energetic relationships and our ability accurately to predict binding affinities.

Hydration and convergence temperatures: on the use and interpretation of correlation plots

An understanding of the energetics of hydration of protein functional groups is essential to understanding the stability and folding of proteins. Much can be learned about hydration energetics by the study of the transfer of model compounds into water. An important feature, common to model compound dissolution and to protein unfolding, is the presence of convergence temperatures at which ΔS°(orΔH°) for each compound in a series takes on a common value. Here we review the relationship between convergence and group additivity. Analysis of the aqueous dissolution of gaseous alcohols and alkanes shows a large negative entropy change for the alcohols relative to the alkanes. While this has been taken as leading to entropic stabilization of hydrogen bonding in proteins, it is shown that this negative ΔS° arises from changes in internal degrees of freedom and should not be applied to the analysis of protein energetics.

Isothermal Titration Calorimetry

Isothermal titration calorimetry is an ideal technique for measuring biological binding interactions. It does not rely on the presence of chromophores or fluorophores, nor does it require an enzymatic assay. Because the technique relies only on the detection of a heat effect upon binding, it can be used to measure the binding constant, K, the enthalpy of binding, DeltaH degrees and the stoichiometry, or number of binding sites, n. This chapter describes instrumentation, experimental design, and the theoretical underpinnings necessary to run and analyze a calorimetric binding experiment.

Anion binding to a protein–protein complex lacks dependence on net charge

The binding of anions to proteins occurs in numerous physiological and metabolic processes. In an effort to understand the factors important in these interactions, we have studied the weak binding of phosphate and sulfate to a protein–protein complex using isothermal titration calorimetry. To our knowledge, this is the first system in which the thermodynamics of anion binding have been determined calorimetrically. By studying both phosphate and sulfate binding and using a range of pH values, the charge on the anion was varied from ∼ −1 to −2. Surprisingly, no dependence of the binding energetics on the charge of the anion was observed. This result indicates that charge–charge interactions are not the dominant factor in binding and suggests the importance of hydrogen bonding in specifically recognizing and coordinating anions.

Thermodynamics of the helix‐coil transition: Binding of S15 and a hybrid sequence, disulfide stabilized peptide to the S‐protein

Pancreatic ribonuclease A may be cleaved to produce two fragments: the S‐peptide (residues 1–20) and the S‐protein (residues 21–124). The S‐peptide, or a truncated version designated as the S15 peptide (residues 1–15), combines with the S‐protein to produce catalytically active complexes. The conformation of these peptides and many of their analogues is predominantly random coil at room temperature; however, they populate a significant fraction of helical form at low temperature under certain solution conditions. Moreover, they adopt a helical conformation when bound to the S‐protein. A hybrid sequence, disulfide‐stabilized peptide (ApaS‐25), designed to stabilize the helical structure of the S‐peptide in solution, also combines with the S‐protein to yield a catalytically active complex. We have performed high‐precision titration microcalorimetric measurements to determine the free energy, enthalpy, entropy, and heat capacity changes for the binding of ApaS‐25 to S‐protein within the temperature range 5–25°C. The thermodynamic parameters for both the complex formation reactions and the helix‐to‐coil transition also were calculated, using a structure‐based approach, by calculating changes in accessible surface area and using published empirical parameters. A simple thermodynamic model is presented in an attempt to account for the differences between the binding of ApaS‐25 and the S‐peptide. From this model, the thermodynamic parameters of the helix‐to‐coil transition of S15 can be calculated. Proteins 2001;42:523–530.