Hydration differences between the major and minor grooves of DNA revealed from heat capacity measurements.

The nature of water on the surface of a macromolecule is reflected in the temperature dependence of the heat effect, i.e., the heat capacity change, ΔCp, that accompanies its removal on forming a complex. The relationship between ΔCp and the nature of the surface dehydrated cannot be modeled for DNA by the use of small molecules, as previously done for proteins, since the contiguous surfaces of the grooves cannot be treated as the sum of small component molecules such as nucleotides. An alternative approach is used here in which ΔCp is measured for the formation of several protein/DNA complexes and the calculated contribution from protein dehydration subtracted to yield the heat capacity change attributable to dehydration of the DNA. The polar and apolar surface areas of the DNA dehydrated on complex formation were calculated from the known structures of the complexes, allowing heat capacity coefficients to be derived representing dehydration of unit surface area of polar and apolar surface in both grooves. Dehydration of apolar surfaces in both grooves is essentially identical and accompanied by a reduction in ΔCp by about 3 J K-1 mol-1 (Å2)-1, a value of somewhat greater magnitude than observed for proteins {ΔCp = - 1.79 J K-1 mol-1 (Å2)-1}. In contrast, dehydration of polar surfaces is very different in the two grooves: in the minor groove ΔCp increases by 2.7 J K-1 mol-1 (Å2)-1, but in the major groove, although ΔCp is also positive, it is low in value: + 0.4 J K-1 mol-1 (Å2)-1. Physical explanations for the magnitudes of ΔCp are discussed.

Conformation and Dynamics of Human Urotensin II and Urotensin Related Peptide in Aqueous Solution.

Conformation and dynamics of the vasoconstrictive peptides human urotensin II (UII) and urotensin related peptide (URP) have been investigated by both unrestrained and enhanced-sampling molecular-dynamics (MD) simulations and NMR spectroscopy. These peptides are natural ligands of the G-protein coupled urotensin II receptor (UTR) and have been linked to mammalian pathophysiology. UII and URP cannot be characterized by a single structure but exist as an equilibrium of two main classes of ring conformations, open and folded, with rapidly interchanging subtypes. The open states are characterized by turns of various types centered at K8Y9 or F6W7 predominantly with no or only sparsely populated transannular hydrogen bonds. The folded conformations show multiple turns stabilized by highly populated transannular hydrogen bonds comprising centers F6W7K8 or W7K8Y9. Some of these conformations have not been characterized previously. The equilibrium populations that are experimentally difficult to access were estimated by replica-exchange MD simulations and validated by comparison of experimental NMR data with chemical shifts calculated with density-functional theory. UII exhibits approximately 72% open:28% folded conformations in aqueous solution. URP shows very similar ring conformations as UII but differs in an open:folded equilibrium shifted further toward open conformations (86:14) possibly arising from the absence of folded N-terminal tail-ring interaction. The results suggest that the different biological effects of UII and URP are not caused by differences in ring conformations but rather by different interactions with UTR.

Enthalpy-entropy compensation: the role of solvation.

Structural modifications to interacting systems frequently lead to changes in both the enthalpy (heat) and entropy of the process that compensate each other, so that the Gibbs free energy is little changed: a major barrier to the development of lead compounds in drug discovery. The conventional explanation for such enthalpy-entropy compensation (EEC) is that tighter contacts lead to a more negative enthalpy but increased molecular constraints, i.e., a compensating conformational entropy reduction. Changes in solvation can also contribute to EEC but this contribution is infrequently discussed. We review long-established and recent cases of EEC and conclude that the large fluctuations in enthalpy and entropy observed are too great to be a result of only conformational changes and must result, to a considerable degree, from variations in the amounts of water immobilized or released on forming complexes. Two systems exhibiting EEC show a correlation between calorimetric entropies and local mobilities, interpreted to mean conformational control of the binding entropy/free energy. However, a substantial contribution from solvation gives the same effect, as a consequence of a structural link between the amount of bound water and the protein flexibility. Only by assuming substantial changes in solvation-an intrinsically compensatory process-can a more complete understanding of EEC be obtained. Faced with such large, and compensating, changes in the enthalpies and entropies of binding, the best approach to engineering elevated affinities must be through the addition of ionic links, as they generate increased entropy without affecting the enthalpy.

Can Simulations and Modeling Decipher NMR Data for Conformational Equilibria? Arginine-Vasopressin.

Arginine vasopressin (AVP) has been suggested by molecular-dynamics (MD) simulations to exist as a mixture of conformations in solution. The (1)H and (13)C NMR chemical shifts of AVP in solution have been calculated for this conformational ensemble of ring conformations (identified from a 23 µs molecular-dynamics simulation). The relative free energies of these conformations were calculated using classical metadynamics simulations in explicit water. Chemical shifts for representative conformations were calculated using density-functional theory. Comparison with experiment and analysis of the results suggests that the (1)H chemical shifts are most useful for assigning equilibrium concentrations of the conformations in this case. (13)C chemical shifts distinguish less clearly between conformations, and the distances calculated from the nuclear Overhauser effect do not allow the conformations to be assigned clearly. The (1)H chemical shifts can be reproduced with a standard error of less than 0.24 ppm (<2.2 ppm for (13)C). The combined experimental and theoretical results suggest that AVP exists in an equilibrium of approximately 70% saddlelike and 30% clinched open conformations. Both newly introduced statistical metrics designed to judge the significance of the results and Smith and Goodman's DP4 probabilities are presented.

The interface between catalytic and hemopexin domains in matrix metalloproteinase-1 conceals a collagen binding exosite.

Matrix metalloproteinase-1 (MMP-1) is an instigator of collagenolysis, the catabolism of triple helical collagen. Previous studies have implicated its hemopexin (HPX) domain in binding and possibly destabilizing the collagen substrate in preparation for hydrolysis of the polypeptide backbone by the catalytic (CAT) domain. Here, we use biophysical methods to study the complex formed between the MMP-1 HPX domain and a synthetic triple helical peptide (THP) that encompasses the MMP-1 cleavage site of the collagen α1(I) chain. The two components interact with 1:1 stoichiometry and micromolar affinity via a binding site within blades 1 and 2 of the four-bladed HPX domain propeller. Subsequent site-directed mutagenesis and assay implicates blade 1 residues Phe(301), Val(319), and Asp(338) in collagen binding. Intriguingly, Phe(301) is partially masked by the CAT domain in the crystal structure of full-length MMP-1 implying that transient separation of the domains is important in collagen recognition. However, mutation of this residue in the intact enzyme disrupts the CAT-HPX interface resulting in a drastic decrease in binding activity. Thus, a balanced equilibrium between these compact and dislocated states may be an essential feature of MMP-1 collagenase activity.

Design and structure of stapled peptides binding to estrogen receptors.

Synthetic peptides that specifically bind nuclear hormone receptors offer an alternative approach to small molecules for the modulation of receptor signaling and subsequent gene expression. Here we describe the design of a series of novel stapled peptides that bind the coactivator peptide site of estrogen receptors. Using a number of biophysical techniques, including crystal structure analysis of receptor-stapled peptide complexes, we describe in detail the molecular interactions and demonstrate that all-hydrocarbon staples modulate molecular recognition events. The findings have implications for the design of stapled peptides in general.

Fluorescence spectroscopy and anisotropy in the analysis of DNA-protein interactions.

Fluorescence spectroscopy can be used as a sensitive non-destructive technique for the characterisation of protein-DNA interactions. A comparison of the intrinsic emission spectra obtained for a protein-DNA complex and for free protein can be informative about the environment of tryptophan and tyrosine residues in the two states. Often there is quenching of the fluorescence intensity of an intrinsic emission spectrum and/or a shift in the wavelength maximum on protein binding to DNA. A step-by-step protocol describes the determination of a DNA-binding curve by measurement of the quenching of the intrinsic protein fluorescence.Fluorescence anisotropy can also be used to obtain a DNA-binding curve if the molecular size of the protein-DNA complex is sufficiently different from the free fluorescing component. Typically an extrinsic fluorophore attached to one or both 5' ends of single-stranded or duplex DNA is used, for this increases the sensitivity of measurement.Fitting of the binding curves, assuming a model, can often yield the stoichiometry and association constant of the interaction. The approach is illustrated using the interaction of the DNA-binding domains (HMG boxes) of mouse Sox-5 and mammalian HMGB1 with short DNA duplexes.

Defining the thermodynamics of protein/DNA complexes and their components using micro-calorimetry.

Understanding the forces driving formation of protein/DNA complexes requires measurement of the Gibbs energy of association, DeltaG, and its component enthalpic, DeltaH, and entropic, DeltaS, contributions. Isothermal titration calorimetry provides the enthalpy (heat) of the binding reaction and an estimate of the association constant, if not too high. Repeating the ITC experiment at several temperatures yields DeltaC ( p ), the change in heat capacity, an important quantity permitting extrapolation of enthalpies and entropies to temperatures outside the experimental range. Binding constants, i.e. Gibbs energies, are best obtained by optical methods such as fluorescence at temperatures where the components are maximally folded. Since DNA-binding domains are often partially unfolded at physiological temperatures, the ITC-observed enthalpy of binding may need to be corrected for the negative contribution from protein refolding. This correction is obtained by differential scanning calorimetric melting of the free DNA-binding domain. Corrected enthalpies are finally combined with accurate Gibbs energies to yield the entropy factor (TDeltaS) at various temperatures. Gibbs energies can be separated into electrostatic and non-electrostatic contributions from the ionic strength dependence of the binding constant.

DNA binding and bending by HMG boxes: energetic determinants of specificity.

To clarify the physical basis of DNA binding specificity, the thermodynamic properties and DNA binding and bending abilities of the DNA binding domains (DBDs) of sequence-specific (SS) and non-sequence-specific (NSS) HMG box proteins were studied with various DNA recognition sequences using micro-calorimetric and optical methods. Temperature-induced unfolding of the free DBDs showed that their structure does not represent a single cooperative unit but is subdivided into two (in the case of NSS DBDs) or three (in the case of SS DBDs) sub-domains, which differ in stability. Both types of HMG box, most particularly SS, are partially unfolded even at room temperature but association with DNA results in stabilization and cooperation of all the sub-domains. Binding and bending measurements using fluorescence spectroscopy over a range of ionic strengths, combined with calorimetric data, allowed separation of the electrostatic and non-electrostatic components of the Gibbs energies of DNA binding, yielding their enthalpic and entropic terms and an estimate of their contributions to DNA binding and bending. In all cases electrostatic interactions dominate non-electrostatic in the association of a DBD with DNA. The main difference between SS and NSS complexes is that SS are formed with an enthalpy close to zero and a negative heat capacity effect, while NSS are formed with a very positive enthalpy and a positive heat capacity effect. This indicates that formation of SS HMG box-DNA complexes is specified by extensive van der Waals contacts between apolar groups, i.e. a more tightly packed interface forms than in NSS complexes. The other principal difference is that DNA bending by the NSS DBDs is driven almost entirely by the electrostatic component of the binding energy, while DNA bending by SS DBDs is driven mainly by the non-electrostatic component. The basic extensions of both categories of HMG box play a similar role in DNA binding and bending, making solely electrostatic interactions with the DNA.

Structure and RNA interactions of the N-terminal RRM domains of PTB.

The polypyrimidine tract binding protein (PTB) is an important regulator of alternative splicing that also affects mRNA localization, stabilization, polyadenylation, and translation. NMR structural analysis of the N-terminal half of PTB (residues 55-301) shows a canonical structure for RRM1 but reveals novel extensions to the beta strands and C terminus of RRM2 that significantly modify the beta sheet RNA binding surface. Although PTB contains four RNA recognition motifs (RRMs), it is widely held that only RRMs 3 and 4 are involved in RNA binding and that RRM2 mediates homodimerization. However, we show here not only that the RRMs 1 and 2 contribute substantially to RNA binding but also that full-length PTB is monomeric, with an elongated structure determined by X-ray solution scattering that is consistent with a linear arrangement of the constituent RRMs. These new insights into the structure and RNA binding properties of PTB suggest revised models of its mechanism of action.