Conformational Entropy Change Research Articles

Introducing NMR strategies to define water molecules that drive metal binding in a transcriptional regulator

Staphylococcus aureus CzrA is a paradigmatic member of the ArsR family of transcriptional metalloregulators, which are critical for the bacterial response to stress. Zinc binding to CzrA, which induces DNA derepression, is entropically driven, as shown by calorimetry. A detailed equilibrium dynamics study of different allosteric states of CzrA revealed that zinc induces an entropy redistribution that controls for DNA binding regulation; however, this change in conformational entropy only accounts for a small net contribution to the total entropy. This difference between the change in conformational entropy vs. total entropy of zinc binding implies a significant contribution of solvent molecule rearrangements to this equilibrium. However, the absence of major structural changes suggests that solvent rearrangements occur mainly on the protein surface and/or from zinc desolvation, concomitant with a dynamical redistribution of conformational entropy. Previous results also suggest that zinc binding not only leads to a redistribution of protein internal dynamics, but also release of water molecules from the protein surface. In turn, these water molecules may make a significant contribution to the allosteric response that results in dissociation from the DNA.Quantifying the differential hydration of two conformational states that share very similar crystal structures and then correlating this with the protein's solvent entropy change constitutes an unresolved problem, even when thermodynamics suggest a significant contribution of solvent entropy. Here, we present different avenues to dissect hydration dynamics in a metal-binding transcriptional regulator that provide different insights into this complex problem. We explore primary solution NMR tools for probing protein–water interactions: the laboratory frame nuclear Overhauser effect (NOE) and its rotating frame counterpart (ROE) between long-lived water molecules and the protein residues. The wNOE/wROE ratio is a promising tool for the detection of hydration dynamics near the surface of a protein in a site-specific manner, minimizing contamination from bulk solvent. Molecular dynamics simulations and computational methods designed to provide a spatially resolved picture of solvent thermodynamics were also employed to provide a more complete panorama of solvent redistribution.

Effects of Conformational Constraint on Peptide Solubility Limits.

Liquid-liquid phase separation of proteins preferentially involves intrinsically disordered proteins or disordered regions. Understanding the solution chemistry of these phase separations is key to learning how to quantify and manipulate systems that involve such processes. Here, we investigate the effect of cyclization on the liquid-liquid phase separation of short polyglycine peptides. We simulated separate aqueous systems of supersaturated cyclic and linear GGGGG and observed spontaneous liquid-liquid phase separation in each of the solutions. The cyclic GGGGG phase separates less robustly than linear GGGGG and has a higher aqueous solubility, even though linear GGGGG has a more favorable single molecule solvation free energy. The versatile and abundant interpeptide contacts formed by the linear GGGGG stabilize the condensed droplet phase, driving the phase separation in this system. In particular, we find that van der Waals close contact interactions are enriched in the droplet phase as opposed to electrostatic interactions. An analysis of the change in backbone conformational entropy that accompanies the phase transition revealed that cyclic peptides lose significantly less entropy in this process as expected. However, we find that the enhanced interaction enthalpy of linear GGGGG in the droplet phase is enough to compensate for a larger decrease in conformational entropy.

Microscopic diffusion in cationic vesicles across different phases

Understanding the phase behavior and microscopic diffusion mechanism of cationic vesicles is crucial for controlled-release kinetics in gene/DNA transfection. Here, we report our findings on the structure, phase behavior, and microscopic dynamics of a vesicle based on a cationic double-chained surfactant, dihexadecyldimethylammonium bromide (DHDAB), which is a promising candidate for a nonviral gene/DNA carrier. Small-angle neutron-scattering measurements reveal that DHDAB aggregates are in the form of unilamellar vesicles. Calorimetric studies show a strong thermal hysteresis in the heating and cooling cycles following different pathways. Two first-order transitions are observed at 303 and 318 K in the heating cycle. Fourier transform infrared spectroscopy reveals that the first transition (at 303 K) is associated with a polymorphic (solid-solid) transition from coagel to an intermediate crystalline (IC) phase, and subsequently the second transition (at 318 K) is an order-disorder transition from IC to the fluid phase. Interestingly, it is found that in the cooling cycle, the fluid phase directly transforms back into the coagel phase without passing through the IC phase. This is also confirmed by an elastic fixed window scan (EFWS) using incoherent neutron scattering. The phase behavior of DHDAB is in strong contrast to that of its longer-chain counterpart, dioctadecyldimethylammonium bromide (DODAB), where the intermediate gel phase was observed during the cooling cycle. Moreover, no polymorphic transition was observed in the heating cycle of DODAB, in which the coagel phase directly converts to the fluid phase. These sharply contrasting phase behaviors for DHDAB and DODAB suggest the strong dependence of membrane phase behavior to the alkyl chain length. EFWS measurements reveal that the phase transitions observed in the DHDAB membrane are associated with strong changes in the DHDAB dynamics. The changes in conformational entropy of DHDAB molecules estimated from EFWS confirm that the coagel-fluid and IC-fluid phase transitions are accompanied by strong changes in the conformations of the alkyl chains. The microscopic dynamics of DHDAB molecules in each of these phases is investigated by employing quasielastic neutron-scattering (QENS). In the ordered phases (coagel and IC), only localized internal dynamics of the DHDAB is observed in the QENS spectra. However, in the fluid phase, the presence of a long-range lateral diffusion is detected in addition to the localized internal dynamics of the alkyl chain. The lateral motion is found to follow Fickian diffusion. The internal dynamics of DHDAB depends on the phase state of the bilayer. In the ordered phase, the internal dynamics is described by a fractional uniaxial rotational diffusion model. However, in the fluid phase, where alkyl tails are disordered with significant gauche defects, the internal dynamics is described using localized translation within confined spheres. This study highlights the rich phase behavior of the DHDAB membrane and the surfactant dynamics across these phases.

Optoplasmonic Modulation of Cell Metabolic State Promotes Rapid Cell Differentiation.

Cell differentiation plays a vital role in mediating organ formation and tissue repair and regeneration. Although rapid and effective methods to stimulate cell differentiation for clinical purposes are highly desired, it remains a great challenge in the medical fields. Herein, a highly effective and conceptual optical method was developed based on a plasmonic chip platform (made of 2D AuNPs nanomembranes). through effective light-augmented plasmonic regulation of cellular bioenergetics (CBE) and an entropy effect at bionano interfaces, to promote rapid cell differentiation. Compared with traditional methods, the developed optoplasmonic method greatly shortens cell differentiation time from usually more than 10 days to only about 3 days. Upon the optoplasmonic treatment of cells, the conformational and vibration entropy changes of cell membranes were clearly revealed through theoretical simulation and fingerprint spectra of cell membranes. Meanwhile, during the treatment process, bioenergetics levels of cells were elevated with increasing mitochondrial membrane potential (Δψm), which accelerates cell differentiation and proliferation. The developed optoplasmonic method is highly efficient and easy to implement, provides a new perspective and avenue for cell differentiation and proliferation, and has potential application prospects in accelerating tissue repair and regeneration.

Colossal barocaloric effects with ultralow hysteresis in two-dimensional metal–halide perovskites

Pressure-induced thermal changes in solids—barocaloric effects—can be used to drive cooling cycles that offer a promising alternative to traditional vapor-compression technologies. Efficient barocaloric cooling requires materials that undergo reversible phase transitions with large entropy changes, high sensitivity to hydrostatic pressure, and minimal hysteresis, the combination of which has been challenging to achieve in existing barocaloric materials. Here, we report a new mechanism for achieving colossal barocaloric effects that leverages the large volume and conformational entropy changes of hydrocarbon order–disorder transitions within the organic bilayers of select two-dimensional metal–halide perovskites. Significantly, we show how the confined nature of these order–disorder phase transitions and the synthetic tunability of layered perovskites can be leveraged to reduce phase transition hysteresis through careful control over the inorganic–organic interface. The combination of ultralow hysteresis and high pressure sensitivity leads to colossal reversible isothermal entropy changes (>200 J kg−1 K−1) at record-low pressures (<300 bar).

Allostery in the dynamic coactivator domain KIX occurs through minor conformational micro-states.

The coactivator KIX of CBP uses two binding surfaces to recognize multiple activators and exhibits allostery in ternary complex formation. Activator•coactivator interactions are central to transcriptional regulation, yet the microscopic origins of allostery in dynamic proteins like KIX are largely unknown. Here, we investigate the molecular recognition and allosteric manifestations involved in two KIX ternary systems c-Myb•KIX•MLL and pKID•KIX•MLL. Exploring the hypothesis that binary complex formation prepays an entropic cost for positive cooperativity, we utilize molecular dynamics simulations, side chain methyl order parameters, and differential scanning fluorimetry (DSF) to explore conformational entropy changes in KIX. The protein’s configurational micro-states from structural clustering highlight the utility of protein plasticity in molecular recognition and allostery. We find that apo KIX occupies a wide distribution of lowly-populated configurational states. Each binding partner has its own suite of KIX states that it selects, building a model of molecular recognition fingerprints. Allostery is maximized with MLL pre-binding, which corresponds to the observation of a significant reduction in KIX micro-states observed when MLL binds. With all binding partners, the changes in KIX conformational entropy arise predominantly from changes in the most flexible loop. Likewise, we find that a small molecule and mutations allosterically inhibit/enhance activator binding by tuning loop dynamics, suggesting that loop-targeting chemical probes could be developed to alter KIX•activator interactions. Experimentally capturing KIX stabilization is challenging, particularly because of the disordered nature of particular activators. However, DSF melting curves allow for inference of relative entropic changes that occur across complexes, which we compare to our computed entropy changes using simulation methyl order parameters.

Materials Technology for Heterogeneous Polymer Systems : Part II. Investigation of Motional Heterogeneity in Rubber Vulcanizates

For rubber vulcanizates or crosslinked rubbers, inherent characteristics of rubber elasticity are well known to be mainly controlled by the change in conformational entropy under deformation, causing constrained molecular mobility of rubbery network chains. In this part II of special lecture, 1H-pulsed NMR method is applied to observe the motional heterogeneity in the crosslinked rubbers, related to effects of crosslink density and incorporation of reinforcing carbon black fillers on the tensile failure behaviors under various conditions. Furthermore, as for analysis of thermo-oxidative aging behaviors for the crosslinked rubbers, the relationships between the breaking stretch ratio in tensile measurements, λb, and NMR relaxation properties such as spin-spin relaxation time, T 2, and residual dipolar coupling constant , D res, were discussed to predict the failure properties of small pieces sampled from engineering rubber parts, for which tensile tests could not be easily performed.

Complementary approaches to obtaining thermodynamic parameters from protein ligand systems-challenges and opportunities

Protein ligand interactions play an important role in biology. Increasingly the aim is to understand and influence protein ligand binding. The binding process is heavily influenced by its thermodynamic parameters. In order to understand how the whole system thermodynamics work it is important to characterise the individual contribution of each of the systems components. While the change in conformational entropy of the protein can be determined using QENS complementary methods are necessary in order to characterise all components. This paper will describe the challenges that can occur when combining the different methods, as well as how they can be overcome.

Quantification of Conformational Entropy Unravels Effect of Disordered Flanking Region in Coupled Folding and Binding.

Intrinsic disorder (ID) constitutes a new dimension to the protein structure-function relationship. The ability to undergo conformational changes upon binding is a key property of intrinsically disordered proteins and remains challenging to study using conventional methods. A 1994 paper by R. S. Spolar and M. T. Record presented a thermodynamic approach for estimating changes in conformational entropy based on heat capacity changes, allowing quantification of residues folding upon binding. Here, we adapt the method for studies of intrinsically disordered proteins. We integrate additional data to provide a broader experimental foundation for the underlying relations and, based on >500 protein-protein complexes involving disordered proteins, reassess a key relation between polar and nonpolar surface area changes, previously determined using globular protein folding. We demonstrate the improved suitability of the adapted method to studies of the folded αα-hub domain RST from radical-induced cell death 1, whose interactome is characterized by ID. From extensive thermodynamic data, quantifying the conformational entropy changes upon binding, and comparison to the NMR structure, the adapted method improves accuracy for ID-based studies. Furthermore, we apply the method, in conjunction with NMR, to reveal hitherto undetected effects of interaction-motif context. Thus, inclusion of the disordered context of the DREB2A RST-binding motif induces structuring of the binding motif, resulting in major enthalpy-entropy compensation in the interaction interface. This study, also evaluating additional interactions, demonstrates the strength of the ID-adapted Spolar-Record thermodynamic approach for dissection of structural features of ID-based interactions, easily overlooked in traditional studies, and for translation of these into mechanistic knowledge.

Correlated Response of Protein Side-Chain Fluctuations and Conformational Entropy to Ligand Binding.

The heterogeneous fast side-chain dynamics of proteins plays crucial roles in molecular recognition and binding. Site-specific NMR experiments quantify these motions by measuring the model-free order parameter (Oaxis2) on a scale of 0 (most flexible) to 1 (least flexible) for each methyl-containing residue of proteins. Here, we have examined ligand-induced variations in the fast side-chain dynamics and conformational entropy of calmodulin (CaM) using five different CaM-peptide complexes. Oaxis2 of CaM in the ligand-free (Oaxis,U2) and ligand-bound (Oaxis,B2) states are calculated from molecular dynamics trajectories and conformational energy surfaces obtained using the adaptive biasing force (ABF) method. ΔOaxis2 = Oaxis,B2 - Oaxis,U2 follows a Gaussian-like unimodal distribution whose second moment is a potential indicator of the binding affinity of these complexes. The probability for the binding-induced Oaxis,U2 → Oaxis,B2 transition decreases with increasing magnitude of ΔOaxis2, indicating that large flexibility changes are improbable for side chains of CaM after ligand binding. A linear correlation established between ΔOaxis2 and the conformational entropy change of the protein makes possible the determination of the conformational entropy of binding of protein-ligand complexes. The results not only underscore the functional importance of fast side-chain fluctuations but also highlight key motional and thermodynamic correlates of protein-ligand binding.

Thermodynamic Compensation in Peptides Following Liquid-Liquid Phase Separation.

Liquid-liquid phase separation of proteins often incorporates intrinsically disordered proteins or those with disordered regions. Examining these processes via the entropy change is desirable for establishing a quantitative foundation with which to probe and understand these phase transitions. Of interest is the effect of residue sequence on the entropy of the peptide backbone. In this work we model these systems via all atom simulations of liquid-liquid phase separation of peptides. Systems of supersaturated pentapeptides separate into a peptide-dense liquid droplet phase as well as a dilute (saturated) aqueous phase. An analysis of the change in backbone conformational entropy associated with the phase transition was performed. We examined systems of four different pentapeptides (GGGGG, GGQGG, GGNGG, and GGVGG) in order to explore the effect of sequence variation on the conformational entropy, as well as the effect of side chain variation on the physical characteristics of the droplet phases. We find that the loss of conformational entropy that accompanies aqueous → droplet transitions is more than compensated by a decrease in interaction enthalpy as contributions to the free energy change for the process.

Hydration Thermodynamics of Familial Parkinson's Disease-Linked Mutants of α-Synuclein.

The hydration thermodynamics of different mutants of α-synuclein (α-syn) related to familial Parkinson's disease (PD) is explored using a computational approach that combines both molecular dynamics simulations in water and integral equation theory of molecular liquids. This analysis focuses on the change in conformational entropy, hydration free energy (HFE), and partial molar volume of α-syn upon mutation. The results show that A53T, A30P, E46K, and H50Q mutants aggregate more readily and display increased HFE and less negative interaction volume than the wild-type α-syn. In contrast, an opposite trend is observed for the G51D mutant with a lower experimental aggregation rate. The residuewise decomposition analysis of the HFE highlights that the dehydration/hydration of the hydrophilic residue-rich N- and C-termini of α-syn majorly contributes to the change upon mutation. The hydration shell contributions of different residues to the interaction volume are consistent with its increase/decrease upon mutation. This work shows that both HFE and interaction volume determine the aggregation kinetics of α-syn upon mutation and may serve as an appropriate benchmark for the treatment of PD.

SRLS Analysis of 15N-1H NMR Relaxation from the Protein S100A1: Dynamic Structure, Calcium Binding, and Related Changes in Conformational Entropy.

We report on amide (N-H) NMR relaxation from the protein S100A1 analyzed with the slowly relaxing local structure (SRLS) approach. S100A1 comprises two calcium-binding "EF-hands" (helix-loop-helix motifs) connected by a linker. The dynamic structure of this protein, in both calcium-free and calcium-bound form, is described as the restricted local N-H motion coupled to isotropic protein tumbling. The restrictions are given by a rhombic potential, u (∼10 kT), the local motion by a diffusion tensor with rate constant D2 (∼109 s-1), and principal axis tilted from the N-H bond at angle β (10-20°). This parameter combination provides a physically insightful picture of the dynamic structure of S100A1 from the N-H bond perspective. Calcium binding primarily affects the C-terminal EF-hand, among others slowing down the motion of helices III and IV approximately 10-fold. Overall, it brings about significant changes in the shape of the local potential, u, and the orientation of the local diffusion axis, β. Conformational entropy derived from u makes an unfavorable entropic contribution to the free energy of calcium binding estimated at 8.6 ± 0.5 kJ/mol. The N-terminal EF-hand undergoes moderate changes. These findings provide new insights into the calcium-binding process. The same data were analyzed previously with the extended model-free (EMF) method, which is a simple limit of SRLS. In that interpretation, the protein tumbles anisotropically. Locally, calcium binding increases ordering in the loops of S100A1 and conformational exchange (Rex) in the helices of its N-terminal EF-hand. These are very unusual features. We show that they most likely stem from problematic data-fitting, oversimplifications inherent in EMF, and experimental imperfections. Rex is shown to be mainly a fit parameter. By reanalyzing the experimental data with SRLS, which is largely free of these deficiencies, we obtain-as delineated above-physically-relevant structural, kinetic, geometric, and binding information.

Computational Probing of Temperature-Dependent Unfolding of a Small Globular Protein: From Cold to Heat Denaturation

Fully atomistic replica exchange molecular dynamics simulations are performed to compute the stability curve of a small globular protein as accurately as possible. To investigate the individual roles of the protein and water parts, we compute the conformational entropy change of this protein directly from the simulation ensembles. This entropy calculation enables complete separations of the unfolding changes of enthalpy and the entropy into their own protein and hydration components. From this decomposition, we are able to determine the main thermodynamic factors governing the cold and heat unfolding events: the cold and heat unfolding events are largely driven by the hydration enthalpy gain and the protein conformational entropy gain, respectively. This computational study discloses several temperature-dependent unfolding thermodynamic behaviors of the protein and water compartments and establishes their unique relationship. Upon unfolding, the changes of enthalpy and entropy in the protein part are all positive convex functions of temperature, whereas the equivalent changes in the water part are all negative concave functions of temperature. Hence, these two mutually opposing effects from the protein and water parts dictate the thermodynamics of unfolding. Furthermore, consistent with the temperature-dependent behaviors of the protein part, the changes of the solvent-accessible surface area and the radius of gyration of the protein upon unfolding are also convex functions of temperature. Hence, all these new temperature dependences, combined together, pave the way to unveiling the thermodynamic and structural features of protein denaturation events at various temperature conditions.

Ensemble-Based Analysis of the Dynamic Allostery in the PSD-95 PDZ3 Domain in Relation to the General Variability of PDZ Structures.

PDZ domains are abundant interaction hubs found in a number of different proteins and they exhibit characteristic differences in their structure and ligand specificity. Their internal dynamics have been proposed to contribute to their biological activity via changes in conformational entropy upon ligand binding and allosteric modulation. Here we investigate dynamic structural ensembles of PDZ3 of the postsynaptic protein PSD-95, calculated based on previously published backbone and side-chain S order parameters. We show that there are distinct but interdependent structural rearrangements in PDZ3 upon ligand binding and the presence of the intramolecular allosteric modulator helix 3. We have also compared these rearrangements in PDZ1-2 of PSD-95 and the conformational diversity of an extended set of PDZ domains available in the PDB database. We conclude that although the opening-closing rearrangement, occurring upon ligand binding, is likely a general feature for all PDZ domains, the conformer redistribution upon ligand binding along this mode is domain-dependent. Our findings suggest that the structural and functional diversity of PDZ domains is accompanied by a diversity of internal motional modes and their interdependence.

Protein conformational entropy is not slaved to water

Conformational entropy can be an important element of the thermodynamics of protein functions such as the binding of ligands. The observed role for conformational entropy in modulating molecular recognition by proteins is in opposition to an often-invoked theory for the interaction of protein molecules with solvent water. The “solvent slaving” model predicts that protein motion is strongly coupled to various aspects of water such as bulk solvent viscosity and local hydration shell dynamics. Changes in conformational entropy are manifested in alterations of fast internal side chain motion that is detectable by NMR relaxation. We show here that the fast-internal side chain dynamics of several proteins are unaffected by changes to the hydration layer and bulk water. These observations indicate that the participation of conformational entropy in protein function is not dictated by the interaction of protein molecules and solvent water under the range of conditions normally encountered.

Microscopic insight to specificity of metal ion cofactor in DNA cleavage by restriction endonuclease EcoRV.

Restriction endonucleases protect bacterial cells against bacteriophage infection by cleaving the incoming foreign DNA into fragments. In presence of Mg2+ ions, EcoRV is able to cleave the DNA but not in presence of Ca2+ , although the protein binds to DNA in presence of both metal ions. We make an attempt to understand this difference using conformational thermodynamics. We calculate the changes in conformational free energy and entropy of conformational degrees of freedom, like DNA base pair steps and dihedral angles of protein residues in Mg2+ (A)-EcoRV-DNA complex compared to Ca2+ (S)-EcoRV-DNA complex using all-atom molecular dynamics (MD) trajectories of the complexes. We find that despite conformational stability and order in both complexes, the individual degrees of freedom behave differently in the presence of two different metal ions. The base pairs in cleavage region are highly disordered in Ca2+ (S)-EcoRV-DNA compared to Mg2+ (A)-EcoRV-DNA. One of the acidic residues ASP90, coordinating to the metal ion in the vicinity of the cleavage site, is conformationally destabilized and disordered, while basic residue LYS92 gets conformational stability and order in Ca2+ (S) bound complex than in Mg2+ (A) bound complex. The enhanced fluctuations hinder placement of the metal ion in the vicinity of the scissile phosphate of DNA. Similar loss of conformational stability and order in the cleavage region is observed by the replacement of the metal ion. Considering the placement of the metal ion near scissile phosphate as requirement for cleavage action, our results suggest that the changes in conformational stability and order of the base pair steps and the protein residues lead to cofactor sensitivity of the enzyme. Our method based on fluctuations of microscopic conformational variables can be applied to understand enzyme activities in other protein-DNA systems.

Minimization-Aware Recursive K*: A Novel, Provable Algorithm that Accelerates Ensemble-Based Protein Design and Provably Approximates the Energy Landscape.

Protein design algorithms that model continuous sidechain flexibility and conformational ensembles better approximate the in vitro and in vivo behavior of proteins. The previous state of the art, iMinDEE-A*-K*, computes provable ɛ-approximations to partition functions of protein states (e.g., bound vs. unbound) by computing provable, admissible pairwise-minimized energy lower bounds on protein conformations, and using the A* enumeration algorithm to return a gap-free list of lowest-energy conformations. iMinDEE-A*-K* runs in time sublinear in the number of conformations, but can be trapped in loosely-bounded, low-energy conformational wells containing many conformations with highly similar energies. That is, iMinDEE-A*-K* is unable to exploit the correlation between protein conformation and energy: similar conformations often have similar energy. We introduce two new concepts that exploit this correlation: Minimization-Aware Enumeration and Recursive K*. We combine these two insights into a novel algorithm, Minimization-Aware Recursive K* (MARK*), which tightens bounds not on single conformations, but instead on distinct regions of the conformation space. We compare the performance of iMinDEE-A*-K* versus MARK* by running the Branch and Bound over K* (BBK*) algorithm, which provably returns sequences in order of decreasing K* score, using either iMinDEE-A*-K* or MARK* to approximate partition functions. We show on 200 design problems that MARK* not only enumerates and minimizes vastly fewer conformations than the previous state of the art, but also runs up to 2 orders of magnitude faster. Finally, we show that MARK* not only efficiently approximates the partition function, but also provably approximates the energy landscape. To our knowledge, MARK* is the first algorithm to do so. We use MARK* to analyze the change in energy landscape of the bound and unbound states of an HIV-1 capsid protein C-terminal domain in complex with a camelid VHH, and measure the change in conformational entropy induced by binding. Thus, MARK* both accelerates existing designs and offers new capabilities not possible with previous algorithms.

Polarity of the ATP binding site of the Na+,K+-ATPase, gastric H+,K+-ATPase and sarcoplasmic reticulum Ca2+-ATPase

A fluorescence ratiometric method utilizing the probe eosin Y is presented for estimating the ATP binding site polarity of P-type ATPases in different conformational states. The method has been calibrated by measurements in a series of alcohols and tested using complexation of eosin Y with methyl-β-cyclodextrin. The results obtained with the Na+,K+-, H+,K+- and sarcoplasmic reticulum Ca2+-ATPases indicate that the ATP binding site, to which eosin is known to bind, is significantly more polar in the case of the Na+,K+- and H+,K+-ATPases compared to the Ca2+-ATPase. This result was found to be consistent with docking calculations of eosin with the E2 conformational state of the Na+,K+-ATPase and the Ca2+-ATPase. Fluorescence experiments showed that eosin binds significantly more strongly to the E1 conformation of the Na+,K+-ATPase than the E2 conformation, but in the case of the Ca2+-ATPase both fluorescence experiments and docking calculations showed no significant difference in binding affinity between the two conformations. This result could be due to the fact that, in contrast to the Na+,K+- and H+,K+-ATPases, the E2-E1 transition of the Ca2+-ATPase does not involve the movement of a lysine-rich N-terminal tail which may affect the overall enzyme conformation. Consistent with this hypothesis, the eosin affinity of the E1 conformation of the Na+,K+-ATPase was significantly reduced after N-terminal truncation. It is suggested that changes in conformational entropy of the N-terminal tail of the Na+, K+- and the H+,K+-ATPases during the E2-E1 transition could affect the thermodynamic stability of the E1 conformation and hence its ATP binding affinity.

Characterization of the dynamics and the conformational entropy in the binding between TAZ1 and CTAD-HIF-1\u03b1

The interaction between the C-terminal transactivation domain of HIF-1α (CTAD-HIF-1α) and the transcriptional adapter zinc binding 1 (TAZ1) domain of CREB binding protein participate in the initiation of gene transcription during hypoxia. Unbound CTAD-HIF-1α is disordered but undergoes a disorder-to-order transition upon binding to TAZ1. We have here performed NMR side chain and backbone relaxation studies on TAZ1 and side chain relaxation measurements on CTAD-HIF-1α in order to investigate the role of picosecond to nanosecond dynamics. We find that the internal motions are significantly affected upon binding, both on the side chain and the backbone level. The dynamic response corresponds to a conformational entropy change that contributes substantially to the binding thermodynamics for both binding partners. Furthermore, the conformational entropy change for the well-folded TAZ1 varies upon binding to different IDP targets. We further identify a cluster consisting of side chains in bound TAZ1 and CTAD-HIF-1α that experience extensive dynamics and are part of the binding region that involves the N-terminal end of the LPQL motif in CTAD-HIF-1α; a feature that might have an important role in the termination of the hypoxic response.