Change In Solvent-accessible Surface Area Research Articles

Empirical Model of Solvophobic Interactions in Organic Solvents.

An empirical model was developed to predict organic solvophobic effects using N-phenylimide molecular balances functionalized with non-polar alkyl groups. Solution studies and X-ray crystallography confirmed intramolecular alkyl-alkyl interactions in their folded conformers. The structural modularity of the balances allowed systematic variation of alkyl group lengths. Control balances were instrumental in isolating weak organic solvophobic effects by eliminating framework solvent-solute effects. A 19 F NMR label enabled analysis across 46 deuterated and non-deuterated solvent systems. Linear correlations were observed between organic solvophobic effects and solvent cohesive energy density (ced) as well as changes in solvent-accessible surface areas (SASA). Using these empirical relationships, a model was constructed to predict organic solvophobic interaction energy per unit area for any organic solvent with known ced values. The predicted interaction energies aligned with recent organic solvophobic measurements and literature values for the hydrophobic effect on non-polar surfaces confirmed the model's accuracy and utility.

N-Arylimide Molecular Balances: A Comprehensive Platform for Studying Aromatic Interactions in Solution.

Noncovalent interactions of aromatic surfaces play a key role in many biological processes and in determining the properties and utility of synthetic materials, sensors, and catalysts. However, the study of aromatic interactions has been challenging because these interactions are usually very weak and their trends are modulated by many factors such as structural, electronic, steric, and solvent effects. Recently, N-arylimide molecular balances have emerged as highly versatile and effective platforms for studying aromatic interactions in solution. These molecular balances can accurately measure weak noncovalent interactions in solution via their influence on the folded-unfolded conformational equilibrium. The structure (i.e., size, shape, π-conjugation, and substitution) and nature (i.e., element, charge, and polarity) of the π-surfaces and interacting groups can be readily varied, enabling the study of a wide range of aromatic interactions. These include aromatic stacking, heterocyclic aromatic stacking, and alkyl-π, chalcogen-π, silver-π, halogen-π, substituent-π, and solvent-π interactions. The ability to measure a diverse array of aromatic interactions within a single model system provides a unique perspective and insights as the interaction energies, stability trends, and solvent effects for different types of interactions can be directly compared. Some broad conclusions that have emerged from this comprehensive analysis include: (1) The strongest aromatic interactions involve groups with positive charges such as pyridinium and metal ions which interact with the electrostatically negative π-face of the aromatic surface via cation-π or metal-π interactions. Attractive electrostatic interactions can also form between aromatic surfaces and groups with partial positive charges. (2) Electrostatic interactions involving aromatic surfaces can be switched from repulsive to attractive using electron-withdrawing substituents or heterocycles. These electrostatic trends appear to span many types of aromatic interactions involving a polar group interacting with a π-surface such as halogen-π, chalcogen-π, and carbonyl-π. (3) Nonpolar groups form weak but measurable stabilizing interactions with aromatic surfaces in organic solvents due to favorable dispersion and/or solvophobic effects. A good predictor of the interaction strength is provided by the change in solvent-accessible surface area. (4) Solvent effects modulate the aromatic interactions in the forms of solvophobic effects and competitive solvation, which can be modeled using solvent cohesion density and specific solvent-solute interactions.

Single-Molecule Force Spectroscopy of M-Value Mutants of Staphylococcal Nuclease Indicates Complex Protein Folding Landscape

Point mutations have commonly been utilized to investigate the underlying thermodynamic characteristics of proteins. While denaturant-induced unfolding can provide a quantitative measure for the stability of a protein, interpretation of the results depends on the model with which the equations are built. Derived from the analysis, a m-value relates the susceptibility of a protein to a denaturant and can be used as a proxy to the change in solvent-accessible surface area between the native and denatured states. In the case of the model protein Staphylococcal Nuclease (SNase), many mutants that alter the m-value have been identified. There is a long-standing debate as to whether the mutations favor an intermediate state in the unfolding pathway or perturb the residual structure of the denatured state of the protein. Bulk experiments have historically been used to try to discriminate between the various models, but these methods are insensitive to finer details of the unfolding/folding pathway(s) due to signal averaging. Single-molecule force spectroscopy via optical tweezers in the presence of chemical denaturant offers an alternative approach by following individual trajectories of the unfolding/folding pathway(s). Our force ramp experiments have revealed the presence of an intermediate in both the wild-type and mutants of SNase, which was confirmed in subsequent force clamp experiments. Further experimentation using optical tweezers allowed the determination of unfolding rates and contour length changes associated with the folding/unfolding pathway of SNase.

L-Proline and RNA Duplex m-Value Temperature Dependence.

The temperature dependence of l-proline interactions with the RNA dodecamer duplex surface exposed after unfolding was quantified using thermal and isothermal titration denaturation monitored by uv-absorbance. The m-value quantifying proline interactions with the RNA duplex surface area exposed after unfolding was measured using RNA duplexes with GC content ranging between 17 and 83%. The m-values from thermal denaturation decreased with increasing GC content signifying increasingly favorable proline interactions with the exposed RNA surface area. However, m-values from isothermal titration denaturation at 25.0 °C were independent of GC content and less negative than those from thermal denaturation. The m-value from isothermal titration denaturation for a 50% GC RNA duplex decreased (became more negative) as the temperature increased and was in nearly exact agreement with the m-value from thermal denaturation. Since RNA duplex transition temperatures increased with GC content, the more favorable proline interactions with the high GC content duplex surface area observed from thermal denaturation resulted from the temperature dependence of proline interactions rather than the RNA surface chemical composition. The enthalpy contribution to the m-value was positive and small (indicating a slight increase in duplex unfolding enthalpy with proline) while the entropic contribution to the m-value was positive and increased with temperature. Our results will facilitate proline's use as a probe of solvent accessible surface area changes during biochemical reactions at different reaction temperatures.

Effect of Urea on G-Quadruplex Stability.

G-quadruplexes represent a class of noncanonical nucleic acid structures implicated in transcriptional regulation, cellular function, and disease. An understanding of the forces involved in stabilization and destabilization of the G-quadruplex conformation relative to the duplex or single-stranded conformation is a key to elucidating the biological role of G-quadruplex-based genomic switches and the quest for therapeutic means for controlled induction or suppression of a G-quadruplex at selected genomic loci. Solute-solvent interactions provide a ubiquitous and, in many cases, the determining thermodynamic force in maintaining and modulating the stability of nucleic acids. These interactions involve water as well as water-soluble cosolvents that may be present in the solution or in the crowded environment in the cell. We present here the first quantitative investigation of the effect of urea, a destabilizing cosolvent, on the conformational preferences of a G-quadruplex formed by the telomeric d[A(G3T2A)3G3] sequence (Tel22). At 20 mM NaCl and room temperature, Tel22 undergoes a two-state urea-induced unfolding transition. An increase in salt mitigates the deleterious effect of urea on Tel22. The urea m-value of Tel22 normalized per change in solvent-accessible surface area, ΔSA, is similar to those for other DNA and RNA structures while being several-fold larger than that of proteins. Our results suggest that urea can be employed as an analytical tool in thermodynamic characterizations of G-quadruplexes in a manner similar to the use of urea in protein studies. We emphasize the need for further studies involving a larger selection of G-quadruplexes varying in sequence, topology (parallel, antiparallel, hybrid), and molecularity (monomolecular, bimolecular, tetramolecular) to outline the advantages and the limits of the use of urea in G-quadruplex studies. A deeper understanding of the effect of solvent and cosolvents on the differential stability of the G-quadruplex and duplex conformations is a step toward elucidation of the modulating influence of different types of cosolvents on duplex-G-quadruplex molecular switches triggering genomic events.

Temperature Dependence of L-Proline RNA Duplex Destabilization

L-Proline, an osmoprotectant and non-essential amino acid, destabilizes the secondary and tertiary structures of nucleic acids. L-proline interactions with the surface area exposed in unfolding RNA duplexes were quantified using m-values for ten RNA dodecamer duplexes that varied in GC content from 17% to 83%. The RNA dodecamer unfolding transitions were facilitated by thermal denaturation and isothermal titration with proline, both monitored using ultraviolet spectroscopy. The m-values from isothermal titration at 22 °C were independent of GC content. However, m-values from thermal denaturation were greater in magnitude and more negative for higher GC content RNA indicating more favorable proline interactions with the accessible surface area exposed in unfolding high GC content duplexes. Since RNA duplex transition temperatures increase with GC content, the more favorable L-proline interactions with the high GC content duplex surface area result from the temperature dependence of L-proline interactions rather than the RNA surface chemical composition. The enthalpy contribution to the m-value is positive and small (indicating a slight increase in duplex unfolding enthalpy with L-proline) while the entropic contribution to the m-value is positive and increases with temperature. Our results help facilitate using L-proline as a probe of solvent accessible surface area changes during biochemical reactions at different reaction temperatures.

Kinetic evidence for a two-stage mechanism of protein denaturation by guanidinium chloride.

Dry molten globular (DMG) intermediates, an expanded form of the native protein with a dry core, have been observed during denaturant-induced unfolding of many proteins. These observations are counterintuitive because traditional models of chemical denaturation rely on changes in solvent-accessible surface area, and there is no notable change in solvent-accessible surface area during the formation of the DMG. Here we show, using multisite fluorescence resonance energy transfer, far-UV CD, and kinetic thiol-labeling experiments, that the guanidinium chloride (GdmCl)-induced unfolding of RNase H also begins with the formation of the DMG. Population of the DMG occurs within the 5-ms dead time of our measurements. We observe that the size and/or population of the DMG is linearly dependent on [GdmCl], although not as strongly as the second and major step of unfolding, which is accompanied by core solvation and global unfolding. This rapid GdmCl-dependent population of the DMG indicates that GdmCl can interact with the protein before disrupting the hydrophobic core. These results imply that the effect of chemical denaturants cannot be interpreted solely as a disruption of the hydrophobic effect and strongly support recent computational studies, which hypothesize that chemical denaturants first interact directly with the protein surface before completely unfolding the protein in the second step (direct interaction mechanism).

An implementation of hydrophobic force in implicit solvent molecular dynamics simulation for packed proteins

MD simulations of five proteins in which helical chains are held together by hydrophobic packing were carried out to investigate the effect of hydrophobic force on simulated structures of these protein complexes in implicit generalized Born (GB) model. The simulation study employed three different methods to treat hydrophobic effect: the standard GB method that does not include explicit hydrophobic force, the LCPO method that includes explicit hydrophobic force based directly on solvent accessible surface area (SASA), and a proposed packing enforced GB (PEGB) method that includes explicit hydrophobic force based on the radius of gyration of the protein complex. Our simulation study showed that all five protein complexes were unpacked in the standard GB simulation (without explicit hydrophobic force). In the LCPO method, three of the five protein systems remained well packed during the simulation, indicating the need for an explicit hydrophobic force in GB model for these packed protein systems. However, two of the five systems were still unpacked during LCPO simulation. For comparison, all five protein systems remain well packed in simulation using the new PEGB method. Analysis shows that the failure of the LCPO method in two cases is related to the way that SASA changes during the unpacking process for these two systems. These examples showed that standard GB method without explicit hydrophobic force is not suitable for MD simulation of protein systems involving hydrophobic packing. A similar problem remains but to a much lesser extent in the LCPO method for some packed protein systems. The proposed PEGB method seems quite promising for MD simulation of large, multi-domain packed proteins in implicit solvent model.

Cutting Off Functional Loops from Homodimeric Enzyme Superoxide Dismutase 1 (SOD1) Leaves Monomeric β-Barrels

Demetallation of the homodimeric enzyme Cu/Zn-superoxide dismutase (SOD1) is known to unleash pronounced dynamic motions in the long active-site loops that comprise almost a third of the folded structure. The resulting apo species, which shows increased propensity to aggregate, stands out as the prime disease precursor in amyotrophic lateral sclerosis (ALS). Even so, the detailed structural properties of the apoSOD1 framework have remained elusive and controversial. In this study, we examine the structural interplay between the central apoSOD1 barrel and the active-site loops by simply cutting them off; loops IV and VII were substituted with short Gly-Ala-Gly linkers. The results show that loop removal breaks the dimer interface and leads to soluble, monomeric β-barrels with high structural integrity. NMR-detected nuclear Overhauser effects are found between all of the constituent β-strands, confirming ordered interactions across the whole barrel. Moreover, the breathing motions of the SOD1 barrel are overall insensitive to loop removal and yield hydrogen/deuterium protection factors typical for cooperatively folded proteins (i.e. the active-site loops act as a "bolt-on" domain with little dynamic influence on its structural foundation). The sole exceptions are the relatively low protection factors in β-strand 5 and the turn around Gly-93, a hot spot for ALS-provoking mutations, which decrease even further upon loop removal. Taken together, these data suggest that the cytotoxic function of apoSOD1 does not emerge from its folded ground state but from a high energy intermediate or even from the denatured ensemble.

Pressure Perturbation Calorimetry and the Thermodynamics of Noncovalent Interactions in Water: Comparison of Protein−Protein, Protein−Ligand, and Cyclodextrin−Adamantane Complexes

Pressure perturbation calorimetry measurements on a range of cyclodextrin-adamantane, protein-ligand (lysozyme-(GlcNac)(3) and ribonuclease-2'CMP) and protein-protein (cytochrome c peroxidase-pseudoazurin) complexes in aqueous solution show consistent reductions in thermal expansibilities compared to the uncomplexed molecules. Thermodynamic data for binding, obtained by titration calorimetry, are also reported. Changes in molar expansibilities can be related to the decrease in solvation during complexation. Although reasonable estimates for numbers of displaced water molecules may be obtained in the case of rigid cyclodextrin-adamantane complexes, protein expansibility data are less easily reconciled. Comparison of data from this wide range of systems indicates that effects are not simply related to changes in solvent-accessible surface area, but may also involve changes in macromolecular dynamics and flexibility. This adds to the growing consensus that understanding thermodynamic parameters associated with noncovalent interactions requires consideration of changes in internal macromolecular fluctuations and dynamics that may not be related to surface area-related solvation effects alone.

Investigating the Effect of Temperature on Transient Partial Unfolding by Proteolysis

Protein inactivation frequently occurs through partially unfolded states under native conditions, and temperature is an important parameter that affects the susceptibility of proteins to inactivation. While the effect of temperature on global unfolding is well documented, however, experimental characterizations of the temperature effect on partial unfolding are rare. Proteolysis offers a valuable chance to investigate the temperature effect on partial unfolding. By investigating proteolysis kinetics, the energetics of the partially unfolded state responsible for proteolysis (the cleavable state) can be studied. E. coli ribonuclease H (RNase H) has been shown to be cleaved by thermolysin at the amide bond between Thr92 and Ala93 through partial unfolding. Using this cleavage as a model system, we evaluated quantitatively the temperature effect on conformational equilibrium between the native state and a cleavable state. The analysis shows that decrease in temperature from 37 degrees C to 4 degrees C decreases the population in the cleavable state and reduces proteolytic susceptibility of the substrate protein. The conformational change leading to the cleavable state has a temperature-independent positive DeltaH degrees with negligible DeltaC(p) degrees . This thermodynamic characteristic of partial unfolding for proteolysis is quite distinct from that of global unfolding of RNase H that has a considerable DeltaC(p) degrees and a negative DeltaH degrees at low temperature. The distinct thermodynamic characteristics of partial unfolding from global unfolding mainly result from the difference in the changes of solvent-accessible surface area, which confirmed that the temperature effect on partial unfolding is strongly scale-dependent.

QM/MM−PBSA Method To Estimate Free Energies for Reactions in Proteins

We have developed a method to estimate free energies of reactions in proteins, called QM/MM-PBSA. It estimates the internal energy of the reactive site by quantum mechanical (QM) calculations, whereas bonded, electrostatic, and van der Waals interactions with the surrounding protein are calculated at the molecular mechanics (MM) level. The electrostatic part of the solvation energy of the reactant and the product is estimated by solving the Poisson-Boltzmann (PB) equation, and the nonpolar part of the solvation energy is estimated from the change in solvent-accessible surface area (SA). Finally, the change in entropy is estimated from the vibrational frequencies. We test this method for five proton-transfer reactions in the active sites of [Ni,Fe] hydrogenase and copper nitrite reductase. We show that QM/MM-PBSA reproduces the results of a strict QM/MM free-energy perturbation method with a mean absolute deviation (MAD) of 8-10 kJ/mol if snapshots from molecular dynamics simulations are used and 4-14 kJ/mol if a single QM/MM structure is used. This is appreciably better than the original QM/MM results or if the QM energies are supplemented with a point-charge model, a self-consistent reaction field, or a PB model of the protein and the solvent, which give MADs of 22-36 kJ/mol for the same test set.

Energetics of the Human Tel-22 Quadruplex−Telomestatin Interaction: A Molecular Dynamics Study

The formation and stabilization of telomeric quadruplexes has been shown to inhibit the activity of telomerase, thus establishing telomeric DNA quadruplex as an attractive target for cancer therapeutic intervention. In this context, telomestatin, a G-quadruplex-specific ligand known to bind and stabilize G-quadruplex, is of great interest. Knowledge of the three-dimensional structure of telomeric quadruplex and its complex with telomestatin in solution is a prerequisite for structure-based rational drug design. Here, we report the relative stabilities of human telomeric quadruplex (AG3[T2AG3]3) structures under K+ ion conditions and their binding interaction with telomestatin, as determined by molecular dynamics simulations followed by energy calculations. The energetics study shows that, in the presence of K+ ions, mixed hybrid-type Tel-22 quadruplex conformations are more stable than other conformations. The binding free energy for quadruplex-telomestatin interactions suggests that 1:2 binding is favored over 1:1 binding. To further substantiate our results, we also calculated the change in solvent-accessible surface area (DeltaSASA) and heat capacity (DeltaCp) associated with 1:1 and 1:2 binding modes. The extensive investigation performed for quadruplex-telomestatin interaction will assist in understanding the parameters influencing the quadruplex-ligand interaction and will serve as a platform for rational drug design.

FURSMASA: A new approach to rapid scoring functions that uses a MD‐averaged potential energy grid and a solvent‐accessible surface area term with parameters GA fit to experimental data

We demonstrate a new approach to the development of scoring functions through the formulation and parameterization of a new function, which can be used both for rapidly ranking the binding of ligands to proteins and for estimating relative aqueous molecular solubilities. The intent of this work is to introduce a new paradigm for creation of scoring functions, wherein we impose the following criteria upon the function: (1) simple; (2) intuitive; (3) requires no postparameterization tweaking; (4) can be applied (without reparameterization) to multiple target systems; and (5) can be rapidly evaluated for any potential ligand. Following these criteria, a new function, FURSMASA (function for rapid scoring using an MD-averaged grid and the accessible surface area) has been developed. Three novel features of the function include: (1) use of an MD-averaged potential energy grid for ligand-protein interactions, rather than a simple static grid; (2) inclusion of a term that depends on the change in the solvent-accessible surface area changes on an atomic (not molecular) basis; and (3) use of the recently derived predictive index (PI) target when optimizing the function, which focuses the function on its intended purpose of relative ranking. A genetic algorithm is used to optimize the function against test data sets that include ligands for the following proteins: IMPDH, p38, gyrase B, HIV-1, and TACE, as well as the Syracuse Research solubility database. We find that the function is predictive, and can simultaneously fit all the test data sets with cross-validated predictive indices ranging from 0.68 to 0.82. As a test of the ability of this function to predict binding for systems not in the training set, the resulting fitted FURSAMA function is then applied to 23 ligands of the COX-2 enzyme. Comparing the results for COX-2 against those obtained using a variety of well-known rapid scoring functions demonstrates that FURSMASA outperforms all of them in terms of the PI and correlation coefficient. We also find that the FURSAMA function is able to reliably predict the water solubility for 1032 compounds from the Syracuse Research solubility database with a cross-correlated PI of 0.84 and a correlation coefficient R(2) of 0.69. This prediction, which is based solely on a term derived from the atom-based solvent-accessible surface areas, compares favorably with the best prediction methods in the literature, most of which are more complex and/or require experimental data. Finally, as a rigorous test of the applicability to database screening, we apply FURSMASA to large active/decoy ligand databases for IMPDH (400 actives vs. 10,000 decoys), p38 (502 actives vs. 10,000 decoys), and HIV (787 actives vs. 10,000 decoys) used in earlier work to critically evaluate many popular scoring functions, and find that FURSMASA performs surprisingly well for IMPDH and HIV.

Stored Ca2+ Depletion-induced Oligomerization of Stromal Interaction Molecule 1 (STIM1) via the EF-SAM Region

Stromal interaction molecule 1 (STIM1) has recently been identified as a key player in store-operated Ca2+ entry. Endoplasmic reticulum (ER) luminal Ca2+ depletion results in STIM1 redistribution from ER membrane homogeneity to distinctly localized aggregates near the plasma membrane; these changes precede and are linked to cytoplasmic Ca2+ influx via Ca2+ release-activated channels (CRACs). The molecular mechanisms initiating ER STIM1 redistribution and plasma membrane CRAC activity are not well understood. We recombinantly expressed the Ca2+-sensing region of STIM1 consisting of the EF-hand together with the sterile alpha-motif (SAM) domain (EF-SAM) to investigate its Ca2+-related conformational and biochemical features. We demonstrate that Ca2+-loaded EF-SAM (holo) contains high alpha-helicity, whereas EF-SAM in the absence of Ca2+ (apo) is much less compact. Accordingly, the melting temperature (Tm) of the holoform is approximately 25 degrees C higher than apoform; heat and urea-derived thermodynamic parameters indicate a Ca2+-induced stabilization of 3.2 kcal mol(-1). We show that holoEF-SAM exists as a monomer, whereas apoEF-SAM readily forms a dimer and/or oligomer, and that oligomer to monomer transitions and vice versa are at least in part mediated by changes in surface hydrophobicity. Additionally, we find that the Ca2+ binding affinity of EF-SAM is relatively low with an apparent dissociation constant (Kd) of approximately 0.2-0.6 mM and a binding stoichiometry of 1. Our results suggest that EF-SAM actively participates in and is the likely the molecular trigger initiating STIM1 punctae formation via large conformational changes. The low Ca2+ affinity of EF-SAM is reconciled with the confirmed role of STIM1 as an ER Ca2+ sensor.

Energetics in Correlation with Structural Features: The Case of Micellization

Understanding micellization processes at the molecular level has direct relevance for biological self-assembly, folding, and association processes. As such, it requires complete characterization of the micellization thermodynamics, including its correlation with the corresponding structural features. In this context, micellization of a series of model non-ionic surfactants (poly(ethylene glycol) monooctyl ethers, C(8)E(gamma)) was studied by isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). The corresponding structural properties of C(8)E(gamma) micelles were investigated by small-angle X-ray scattering (SAXS). The C(8)E(gamma) micellization, characterized independently from ITC, DSC, and structural data, reveals that deltaH(M)(o) > 0, deltaS(M)(o) > 0, and deltaC(P)(M)(o) < 0, while the dissection of its energetics shows that it is primarily governed by the transfer of 20-30 C(8) alkyl chains from aqueous solution into the nonpolar core (r approximately 1.3 nm) of the spherical micelle. Moreover, thermodynamic parameters of micellization, estimated from the structural features related to the changes in solvent-accessible surface areas upon micellization, are in a good agreement with the corresponding parameters obtained from the analysis of ITC and DSC data. We have shown that the contributions to deltaS(M)(o) other than from hydration (deltaS(M)(other)(o)), estimated from experimental data, appear to be small (deltaS(M)(other)(o) < 0.1 deltaS(M)(other)(o)) and agree well with the theoretical estimates expressed as a sum of the corresponding translational, conformational, and size contributions. These deltaS(M)(other)(o) contributions are much less unfavorable than those estimated for a rigid-body association, which indicates the dynamic nature of the C(8)E(gamma) micellar aggregates. the dynamic nature of the C8EY micellar aggregates.

Structural and Computational Characterization of the SHV-1 β-Lactamase-β-Lactamase Inhibitor Protein Interface

Beta-lactamase inhibitor protein (BLIP) binds a variety of class A beta-lactamases with affinities ranging from micromolar to picomolar. Whereas the TEM-1 and SHV-1 beta-lactamases are almost structurally identical, BLIP binds TEM-1 approximately 1000-fold tighter than SHV-1. Determining the underlying source of this affinity difference is important for understanding the molecular basis of beta-lactamase inhibition and mechanisms of protein-protein interface specificity and affinity. Here we present the 1.6A resolution crystal structure of SHV-1.BLIP. In addition, a point mutation was identified, SHV D104E, that increases SHV.BLIP binding affinity from micromolar to nanomolar. Comparison of the SHV-1.BLIP structure with the published TEM-1.BLIP structure suggests that the increased volume of Glu-104 stabilizes a key binding loop in the interface. Solution of the 1.8A SHV D104K.BLIP crystal structure identifies a novel conformation in which this binding loop is removed from the interface. Using these structural data, we evaluated the ability of EGAD, a program developed for computational protein design, to calculate changes in the stability of mutant beta-lactamase.BLIP complexes. Changes in binding affinity were calculated within an error of 1.6 kcal/mol of the experimental values for 112 mutations at the TEM-1.BLIP interface and within an error of 2.2 kcal/mol for 24 mutations at the SHV-1.BLIP interface. The reasonable success of EGAD in predicting changes in interface stability is a promising step toward understanding the stability of the beta-lactamase.BLIP complexes and computationally assisted design of tight binding BLIP variants.

Physical characterization of MxiH and PrgI, the needle component of the type III secretion apparatus from Shigella and Salmonella

Shigella and Salmonella use similar type III secretion systems for delivering effector proteins into host cells. This secretion system consists of a base anchored in both bacterial membranes and an extracellular "needle" that forms a rod-like structure exposed on the pathogen surface. The needle is composed of multiple subunits of a single protein and makes direct contact with host cells to facilitate protein delivery. The proteins that make up the needle of Shigella and Salmonella are MxiH and PrgI, respectively. These proteins are attractive vaccine candidates because of their essential role in virulence and surface exposure. We therefore isolated, purified, and characterized the monomeric forms of MxiH and PrgI. Their far-UV circular dichroism spectra show structural similarities with hints of subtle differences in their secondary structure. Both proteins are highly helical and thermally unstable, with PrgI having a midpoint of thermal unfolding (Tm) near 37 degrees C and MxiH having a value near 42 degrees C. The two proteins also have comparable intrinsic stabilities as measured by chemically induced (urea) unfolding. MxiH, however, with a free energy of unfolding (DeltaG degrees 0,un) of 1.6 kcal/mol, is slightly more stable than PrgI (1.2 kcal/mol). The relatively low m-values obtained for the urea-induced unfolding of the proteins suggest that they undergo only a small change in solvent-accessible surface area. This argues that when MxiH and PrgI are incorporated into the needle complex, they obtain a more stable structural state through the introduction of protein-protein interactions.

Free Energy Contributions to Direct Readout of a DNA Sequence

The energetic contributions of individual DNA-contacting side chains to specific DNA recognition in the human papillomavirus 16 E2C-DNA complex is small (less than 1.0 kcal mol(-1)), independent of the physical and chemical nature of the interaction, and is strictly additive. The sum of the individual contributions differs 1.0 kcal mol(-1) from the binding energy of the wild-type protein. This difference corresponds to the contribution from the deformability of the DNA, known as "indirect readout." Thus, we can dissect the energetic contribution to DNA binding into 90% direct and 10% indirect readout components. The lack of high energy interactions indicates the absence of "hot spots," such as those found in protein-protein interfaces. These results are compatible with a highly dynamic and "wet" protein-DNA interface, yet highly specific and tight, where individual interactions are constantly being formed and broken.

Energetic diversity of DNA minor-groove recognition by small molecules displayed through some model ligand-DNA systems.

Energetics of interactions occurring in the model ligand-DNA systems constituted from distamycin A (DST), netropsin (NET) and the oligomeric duplexes d(GCAAGTTGCGATATACG)d(CGTATATCGCAACTTGC)=D#1 and d(GCAAGTTGCGAAAAACG)d(CGTTTTTCGCAACTTGC)=D#2 was studied by spectropolarimetry, UV-absorption spectroscopy and isothermal titration calorimetry. Model analysis of the measured signals was applied to describe individual and competitive binding in terms of populations of various species in the solution. Our results reveal several unprecedented ligand-DNA binding features. DST binds to the neighboring 5'-AAGTT-3' and 5'-ATATA-3' sites of D#1 statistically in a 2:1 binding mode. By contrast, its association to D#2 appears to be a 2:1 binding event only at the DST/D#2 molar ratios between 0 and 2 while its further binding to D#2 may be considered as a step-by-step binding to the unoccupied 5'-AAAAA-3' sites resulting first in DST3D#2 and finally in DST4D#2 complex formation. Competition between DST and NET binding shows that for the most part DST displaces NET from its complexes with D#1 and D#2. In contrast to the obligatory 1:1 binding of DST to the ligand-free 5'-AAAAA-3' sites observed at DST/5'-AAAAA-3' <1 the displacement of NET bound to the 5'-AAAAA-3' sites by added DST occurs even at the smallest additions of DST in a 2:1 manner. NET can also displace DST molecules but only those bound monomerically to the 5'-AAAAA-3' sites of DST3D#2. Actually, only half of these molecules can be displaced due to the simultaneous rebinding of the displaced DST to the unreacted 5'-AAAAA-3' sites in DST3D#2. Binding of DST and NET to D#1 and D#2 is an enthalpy driven process accompanied by large unfavorable (DST), small (NET) or large favorable (NET binding to 5'-AAAAA-3') entropy contributions and negative deltaCP degrees that are reasonably close to deltaCP degrees predicted from the calculated changes in solvent-accessible surface areas that accompany complex formation. Although various modes of DST and NET binding within D#1 and D#2 are characterized by significant energetic differences they seem to be governed by the same driving forces; the hydrophobic transfer of ligand from the solution into the duplex binding site and the accompanying specific non-covalent ligand-DNA and ligand-ligand interactions occurring within the DNA minor groove.