Exposure Of Side Chains Research Articles

Laser-free flash oxidation (Fox) hydroxyl radical protein footprinting system accurately maps the paratope and epitope of TNFα bound to adalimumab

The Fox Protein Footprinting System is a novel hydroxyl radical protein footprinting (HRPF) method that uses a proprietary flash oxidation lamp to photolyze hydrogen peroxide to form hydroxyl radicals (⋅OH). The ⋅OH irreversibly modify solvent exposed side chains of amino acids. As solvent accessibility changes, the ⋅OH modification concordantly changes. With the addition of real time radical dosimetry, the effective ·OH concentration can be determined and easily adjusted, allowing conditions with varying scavenging effects to be easily compared.

A Native CPP from Rattlesnake with Therapeutic and Theranostic Properties.

The cell-penetrating peptides (CPPs) are characterized by the ability of internalization into cells in vitro and in vivo, and the ability of these peptides can rely on a high content of positive charges, as it is the case of the native CPP crotamine. Crotamine is a polypeptide of about 42 amino acid residues with high content of basic residues as Arg and Lys. Although most of known CPPs are linear peptides,native crotamine from the venom of a South American rattlesnakehas a well-defined 3D structure stabilized by three disulfide bonds which guarantee the exposure of side chains of basic amino acids. This 3D structure also protects this amphipathic polypeptide from the degradation evenif administered by oral route,therefore, protecting alsothe biological activities of crotamine. As several different biological properties of crotamineare dependent of cell penetration, the methods mainly employed for analyzing crotamine properties as anthelminthic and antimalarial activities, antimicrobial and antitumor activities, with a unique selective cytotoxic property against actively proliferating cells, as tumor cells, were chosen based oncrotamine ability of internalization mediated by its positive charge. This native cationic polypeptide is also able to efficiently carry, with no need of covalent linkage with the cargo, genetic material into cells in vitro and in vivo, suggesting its use in gene therapy. Moreover, the possibility of decorating gold nanoparticles keeping the ability of transfecting cells was demonstrated. More recently, the ability of crotamine to interfere in animal metabolism, inducing browning of adipose tissue and increasing the energy expenditure, and its application in renal therapy was demonstrated. As crotamine also accumulates specifically in tumor cells in vivo, andthe potential utility of crotamine as a theranostic agent was then suggested. Therefore, diverse methodologies employed for the characterization and exploration of the therapeutic applications of this promising native CPP for remediation of several pathogenic conditions are presented here.

Two-step purification of tag-free norovirus-like particles from silkworm larvae (Bombyx mori)

Recombinantly expressed VP1 of norovirus self-assembled and formed norovirus-like particles (NoV-LPs). This native VP1 was expressed using the Bombyx mori nucleopolyhedrovirus (BmNPV) expression system in silkworm larva. NoV-LPs were collected from silkworm fat body lysate by density gradient centrifugation. To improve the purity of the NoV-LP, the proteins were further purified using immobilized metal affinity chromatography based on the surface exposed side chain of histidine residues. The additional purification led to a highly purified virus-like particle (VLP). The morphology and size of the purified VLPs were examined using a transmission electron microscope, and dynamic light scattering revealed a monodispersed spherical morphology with a diameter of 34 nm. The purified product had a purity of >90% with a recovery yield of 48.7% (equivalent to 930 μg) from crude lysate, obtained from seven silkworm larvae. In addition, the purified VLP could be recognized by antibodies against GII norovirus in sandwich enzyme-linked immunosorbent assay, which indicated that the silkworm-derived VLP is biologically functional as a NoV-LP in its native state, is structurally correct, and exerts its biological function. Our results suggest that the silkworm-derived NoV-LP may be useful for subsequent applications, such as in a vaccine platform. Moreover, the silkworm-based expression system is known for its robustness, facile up-scalability, and relatively low expense compared to insect cell systems.

The amyloid concentric β-barrel hypothesis: Models of synuclein oligomers, annular protofibrils, lipoproteins, and transmembrane channels.

Amyloid beta (Aβ of Alzheimer's disease) and α-synuclein (α-Syn of Parkinson's disease) form large fibrils. Evidence is increasing however that much smaller oligomers are more toxic and that these oligomers can form transmembrane ion channels. We have proposed previously that Aβ42 oligomers, annular protofibrils, and ion channels adopt concentric β-barrel molecular structures. Here we extend that hypothesis to the superfamily of α, β, and γ-synucleins. Our models of numerous synuclein oligomers, annular protofibrils, tubular protofibrils, lipoproteins, and ion channels were developed to be consistent with sizes, shapes, molecular weights, and secondary structures of assemblies as determined by electron microscopy and other studies. The models have the following features: (1) all subunits have identical structures and interactions; (2) they are consistent with conventional β-barrel theory; (3) the distance between walls of adjacent β-barrels is between 0.6 and 1.2 nm; (4) hydrogen bonds, salt bridges, interactions among aromatic side-chains, burial and tight packing of hydrophobic side-chains, and aqueous solvent exposure of hydrophilic side-chains are relatively optimal; and (5) residues that are identical among distantly related homologous proteins cluster in the interior of most oligomers whereas residues that are hypervariable are exposed on protein surfaces. Atomic scale models of some assemblies were developed.

Biochemical and biophysical characterisation of a small purified lipase from Rhizopus oryzae ZAC3

The characteristics of a purified lipase from Rhizopus oryzae ZAC3 (RoL-ZAC3) were investigated. RoL-ZAC3, a 15.8 kDa protein, which was optimally active at pH 8 and 55 °C had a half-life of 126 min at 60 °C. The kinetic parameters using p-nitrophenylbutyrate as substrate were 0.19 ± 0.02 mM, 126 ± 5.6 U/ml and 122 s−1 for Km , V max and k cat respectively. RoL-ZAC3 showed stability in methanol and isopropanol with Na+ enhancing the activity. p-nitrophenyloleate and castor oil were the best preferred substrates among the p-nitrophenyl esters and vegetable oils tested respectively. About 43% residual activity was observed after incubation for 30 min at 75 °C. Circular dichroism thermal scan showed that the lipase displayed intense negative ellipticities even at high temperature. Perturbation of the tertiary structure with increasing temperature caused the exposure of hydrophobic side chains to the aqueous environment as revealed by tryptophan fluorescence, with a t−Tm of 50 °C. Differential scanning calorimetry analysis showed melting temperature and calorimetric enthalpy of 55.5 °C and 444 kJ/mol respectively. Dynamic light scattering analysis indicated that the lipase was prone to aggregation upon unfolding at high temperature. It can be concluded that RoL-ZAC3 possesses promising potential for numerous biotechnological applications.

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins.

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. FPOP uses a 248 nm excimer laser to photolyze hydrogen peroxide producing hydroxyl radicals. These radicals oxidatively modify solvent exposed side chains of 19 of the 20 amino acids. Recently, this method has been used in live cells (IC-FPOP) to study protein interactions in their native environment. The study of proteins in cells accounts for intermolecular crowding and various protein interactions that are disrupted for in vitro studies. A custom single cell flow system was designed to reduce cell aggregation and clogging during IC-FPOP. This flow system focuses the cells past the excimer laser individually, thus ensuring consistent irradiation. By comparing the extent of oxidation produced from FPOP to the protein's solvent accessibility calculated from a crystal structure, IC-FPOP can accurately probe the solvent accessible side chains of proteins.

Mutation in a flexible linker modulates binding affinity for modular complexes.

Tandem beta zippers are modular complexes formed between repeated linear motifs and tandemly arrayed domains of partner proteins in which β-strands form upon binding. Studies of such complexes, formed by LIM domain proteins and linear motifs in their intrinsically disordered partners, revealed spacer regions between the linear motifs that are relatively flexible but may affect the overall orientation of the binding modules. We demonstrate that mutation of a solvent exposed side chain in the spacer region of an LHX4-ISL2 complex has no significant effect on the structure of the complex, but decreases binding affinity, apparently by increasing flexibility of the linker.

Interplay of cysteine exposure and global protein dynamics in small-molecule recognition by a regulator of G-protein signaling protein.

Regulator of G protein signaling (RGS) proteins play a pivotal role in regulation of G protein-coupled receptor (GPCR) signaling and are therefore becoming an increasingly important therapeutic target. Recently discovered thiadiazolidinone (TDZD) compounds that target cysteine residues have shown different levels of specificities and potencies for the RGS4 protein, thereby suggesting intrinsic differences in dynamics of this protein upon binding of these compounds. In this work, we investigated using atomistic molecular dynamics (MD) simulations the effect of binding of several small-molecule inhibitors on perturbations and dynamical motions in RGS4. Specifically, we studied two conformational models of RGS4 in which a buried cysteine residue is solvent-exposed due to side-chain motions or due to flexibility in neighboring helices. We found that TDZD compounds with aromatic functional groups perturb the RGS4 structure more than compounds with aliphatic functional groups. Moreover, small-molecules with aromatic functional groups but lacking sulfur atoms only transiently reside within the protein and spontaneously dissociate to the solvent. We further measured inhibitory effects of TDZD compounds using a protein-protein interaction assay on a single-cysteine RGS4 protein showing trends in potencies of compounds consistent with our simulation studies. Thermodynamic analyses of RGS4 conformations in the apo-state and on binding to TDZD compounds revealed links between both conformational models of RGS4. The exposure of cysteine side-chains appears to facilitate initial binding of TDZD compounds followed by migration of the compound into a bundle of four helices, thereby causing allosteric perturbations in the RGS/Gα protein-protein interface.

CpAMs induce assembly of HBV capsids with altered electrophoresis mobility: Implications for mechanism of inhibiting pgRNA packaging

Native agarose gel electrophoresis-based particle gel assay has been commonly used for examination of hepatitis B virus (HBV) capsid assembly and pregenomic RNA encapsidation in HBV replicating cells. Interestingly, treatment of cells with several chemotypes of HBV core protein allosteric modulators (CpAMs) induced the assembly of both empty and DNA-containing capsids with faster electrophoresis mobility. In an effort to determine the physical basis of CpAM-induced capsid mobility shift, we found that the surface charge, but not the size, of capsids is the primary determinant of electrophoresis mobility. Specifically, through alanine scanning mutagenesis analysis of twenty-seven charged amino acids in core protein assembly domain and hinge region, we showed that except for K7 and E8, substitution of glutamine acid (E) or aspartic acid (D) on the surface of capsids reduced their mobility, but substitution of lysine (K) or arginine (R) on the surface of capsids increased their mobility in variable degrees. However, alanine substitution of the charged amino acids that are not exposed on the surface of capsid did not apparently alter capsid mobility. Hence, CpAM-induced electrophoresis mobility shift of capsids may reflect the global alteration of capsid structure that changes the exposure and/or ionization of charged amino acid side chains of core protein. Our findings imply that CpAM inhibition of pgRNA encapsidation is possibly due to the assembly of structurally altered nucleocapsids. Practically, capsid electrophoresis mobility shift is a diagnostic marker of compounds that target core protein assembly and predicts sensitivity of HBV strains to specific CpAMs.

Prediction of delayed retention of antibodies in hydrophobic interaction chromatography from sequence using machine learning.

The hydrophobicity of a monoclonal antibody is an important biophysical property relevant for its developability into a therapeutic. In addition to characterizing heterogeneity, Hydrophobic Interaction Chromatography (HIC) is an assay that is often used to quantify the hydrophobicity of an antibody to assess downstream risks. Earlier studies have shown that retention times in this assay can be correlated to amino-acid or atomic propensities weighted by the surface areas obtained from protein 3-dimensional structures. The goal of this study is to develop models to enable prediction of delayed HIC retention times directly from sequence. We utilize the randomforest machine learning approach to estimate the surface exposure of amino-acid side-chains in the variable region directly from the antibody sequence. We obtain mean-absolute errors of 4.6% for the prediction of surface exposure. Using experimental HIC data along with the estimated surface areas, we derive an amino-acid propensity scale that enables prediction of antibodies likely to have delayed retention times in the assay. We achieve a cross-validation Area Under Curve of 0.85 for the Receiver Operating Characteristic curve of our model. The low computational expense and high accuracy of this approach enables real-time assessment of hydrophobic character to enable prioritization of antibodies during the discovery process and rational engineering to reduce hydrophobic liabilities. Structure data, aligned sequences, experimental data and prediction scores for test-cases, and R scripts used in this work are provided as part of the Supplementary Material. tushar.jain@adimab.com. Supplementary data are available at Bioinformatics online.

Mechanical Deformation Accelerates Protein Ageing.

A hallmark of tissue ageing is the irreversible oxidative modification of its proteins. We show that single proteins, kept unfolded and extended by a mechanical force, undergo accelerated ageing in times scales of minutes to days. A protein forced to be continuously unfolded completely loses its ability to contract by folding, becoming a labile polymer. Ageing rates vary among different proteins, but in all cases they lose their mechanical integrity. Random oxidative modification of cryptic side chains exposed by mechanical unfolding can be slowed by the addition of antioxidants such as ascorbic acid, or accelerated by oxidants. By contrast, proteins kept in the folded state and probed over week-long experiments show greatly reduced rates of ageing. We demonstrate a novel approach whereby protein ageing can be greatly accelerated: the constant unfolding of a protein for hours to days is equivalent to decades of exposure to free radicals under physiological conditions.

Photolytic Cross-Linking to Probe Protein-Protein and Protein-Matrix Interactions in Lyophilized Powders.

Protein structure and local environment in lyophilized formulations were probed using high-resolution solid-state photolytic cross-linking with mass spectrometric analysis (ssPC-MS). In order to characterize structure and microenvironment, protein-protein, protein-excipient, and protein-water interactions in lyophilized powders were identified. Myoglobin (Mb) was derivatized in solution with the heterobifunctional probe succinimidyl 4,4'-azipentanoate (SDA) and the structural integrity of the labeled protein (Mb-SDA) confirmed using CD spectroscopy and liquid chromatography/mass spectrometry (LC-MS). Mb-SDA was then formulated with and without excipients (raffinose, guanidine hydrochloride (Gdn HCl)) and lyophilized. The freeze-dried powder was irradiated with ultraviolet light at 365 nm for 30 min to produce cross-linked adducts that were analyzed at the intact protein level and after trypsin digestion. SDA-labeling produced Mb carrying up to five labels, as detected by LC-MS. Following lyophilization and irradiation, cross-linked peptide-peptide, peptide-water, and peptide-raffinose adducts were detected. The exposure of Mb side chains to the matrix was quantified based on the number of different peptide-peptide, peptide-water, and peptide-excipient adducts detected. In the absence of excipients, peptide-peptide adducts involving the CD, DE, and EF loops and helix H were common. In the raffinose formulation, peptide-peptide adducts were more distributed throughout the molecule. The Gdn HCl formulation showed more protein-protein and protein-water adducts than the other formulations, consistent with protein unfolding and increased matrix interactions. The results demonstrate that ssPC-MS can be used to distinguish excipient effects and characterize the local protein environment in lyophilized formulations with high resolution.

Structure of the iron-free true C-terminal half of bovine lactoferrin produced by tryptic digestion and its functional significance in the gut.

Bovine lactoferrin, a 76-kDa glycoprotein (Ala1-Arg689) consists of two similar N- and C-terminal molecular halves with the ability to bind two Fe(3+) ions. The N-terminal half, designated as the N-lobe (Ala1-Arg341) and the C-terminal half designated as the C-lobe (Tyr342-Arg689) have similar iron-binding properties, but the resistant C-lobe prolongs the physiological role of bovine lactoferrin in the digestive tract. Here, we report the crystal structure of true C-lobe, which was produced by limited proteolysis of bovine lactoferrin using trypsin. In the first proteolysis step, two fragments of 21 kDa (Glu86-Lys282) and 45 kDa (Ser283-Arg689) were generated because two lysine residues, Lys85 and Lys282, in the structure of iron-saturated bovine lactoferrin were fully exposed. The 45-kDa fragment was further digested at the newly exposed side chain of Arg341, generating a 38-kDa perfect C-lobe (Tyr342-Arg689). By contrast, the apo-lactoferrin was cut by trypsin only at Arg341, which was exposed in the structure of apo-lactoferrin, whereas the other two sites with Lys85 and Lys282 are inaccessible. The purified iron-saturated C-lobe was crystallized at pH 4.0. The structure was determined by the molecular replacement method using coordinates of the C-terminal half (Arg342-Arg689) of intact camel apo-lactoferrin. The structure determination revealed that the iron atom was absent and the iron-binding cleft was found in a wide-open conformation, whereas in the previously determined structure of iron-saturated C-lobe of bovine lactoferrin, the iron atom was present and the iron-binding site was in the closed confirmation.

Crystal structure of HIV-1 Tat complexed with human P-TEFb and AFF4

Developing anti-viral therapies targeting HIV-1 transcription has been hampered by the limited structural knowledge of the proteins involved. HIV-1 hijacks the cellular machinery that controls RNA polymerase II elongation through an interaction of HIV-1 Tat with the positive transcription elongation factor P-TEFb, which interacts with an AF4 family member (AFF1/2/3/4) in the super elongation complex (SEC). Because inclusion of Tat•P-TEFb into the SEC is critical for HIV transcription, we have determined the crystal structure of the Tat•AFF4•P-TEFb complex containing HIV-1 Tat (residues 1–48), human Cyclin T1 (1–266), human Cdk9 (7–332), and human AFF4 (27–69). Tat binding to AFF4•P-TEFb causes concerted structural changes in AFF4 via a shift of helix H5′ of Cyclin T1 and the α-310 helix of AFF4. The interaction between Tat and AFF4 provides structural constraints that explain tolerated Tat mutations. Analysis of the Tat-binding surface of AFF4 coupled with modeling of all other AF4 family members suggests that AFF1 and AFF4 would be preferred over AFF2 or AFF3 for interaction with Tat•P-TEFb. The structure establishes that the Tat-TAR recognition motif (TRM) in Cyclin T1 interacts with both Tat and AFF4, leading to the exposure of arginine side chains for binding to TAR RNA. Furthermore, modeling of Tat Lys28 acetylation suggests that the acetyl group would be in a favorable position for H-bond formation with Asn257 of TRM, thereby stabilizing the TRM in Cyclin T1, and provides a structural basis for the modulation of TAR RNA binding by acetylation of Tat Lys28.

A plate-based high-throughput assay for virus stability and vaccine formulation

Standard methods for assessing the thermal stability of viruses can be time consuming and rather qualitative yet such data is a necessary requisite for vaccine formulation. In this study a novel plate-based thermal scanning assay for virus particle stability has been developed (PaSTRy: Particle Stability Thermal Release Assay). Two environment-sensitive fluorescent dyes, with non-overlapping emission spectra and different affinities, are used to accrue simultaneously independent data for the overall stability of the virus capsid, as judged by the exposure of the genome, and for capsid protein stability according to the exposure of hydrophobic side chains which are normally buried. This offers a fast and efficient high-throughput method to optimise vaccine formulation and to investigate the processes of virus uncoating.

Functional and structural basis of T cell recognition in autoimmune gastritis: mapping of autoantigenic peptides of the α-chain of the H/K ATPase and X-ray structure of the I-Ad/peptide complexes (106.25)

Abstract We have previously reported the development of mouse models of autoimmune gastritis in which BALB/c mice expressing transgenic αβ TCR, designated A23 and A51, spontaneously develop acute Th1-mediated high-penetrance and chronic Th2-mediated low penetrance forms of the disease, respectively. Using an array of synthetic mutated peptides in functional and MHC II-binding assays, we now report the core peptides recognized by each of these TCR. A23 recognizes H/K ATPase residues 633-641 (AKAIAASVG) and A51 sees 892-900 (CVGLRPQWE), both exhibiting a canonical 1, 4, 6, 9 I-Ad -binding motif. To extend these findings, we determined the X-ray crystallographic structure of complexes of I-Ad with both covalent and non-covalent peptides that stimulate the A51 TCR, and used these structures to generate a molecular model of the A23-stimulating peptide/I-Ad complex. The structural identification of peptide anchor residues in individual I-Ad binding pockets and of solvent exposed side chains as T cell epitopic residues readily explains T cell proliferation data and I-Ad/peptide off-rate measurements of the large panel of truncated, chimeric, and alanine or glycine-substituted synthetic peptides. The first structure of I-Ad in complex with an autoimmune peptide offers new insight in the general question of the structural basis of responses to self antigens.

Charge Transfer in Model Peptides: Obtaining Marcus Parameters from Molecular Simulation

Charge transfer within and between biomolecules remains a highly active field of biophysics. Due to the complexities of real systems, model compounds are a useful alternative to study the mechanistic fundamentals of charge transfer. In recent years, such model experiments have been underpinned by molecular simulation methods as well. In this work, we study electron hole transfer in helical model peptides by means of molecular dynamics simulations. A theoretical framework to extract Marcus parameters of charge transfer from simulations is presented. We find that the peptides form stable helical structures with sequence dependent small deviations from ideal PPII helices. We identify direct exposure of charged side chains to solvent as a cause of high reorganization energies, significantly larger than typical for electron transfer in proteins. This, together with small direct couplings, makes long-range superexchange electron transport in this system very slow. In good agreement with experiment, direct transfer between the terminal amino acid side chains can be dicounted in favor of a two-step hopping process if appropriate bridging groups exist.

Cell-Cell Membrane Fusion Induced by p15 Fusion-associated Small Transmembrane (FAST) Protein Requires a Novel Fusion Peptide Motif Containing a Myristoylated Polyproline Type II Helix

The p15 fusion-associated small transmembrane (FAST) protein is a nonstructural viral protein that induces cell-cell fusion and syncytium formation. The exceptionally small, myristoylated N-terminal ectodomain of p15 lacks any of the defining features of a typical viral fusion protein. NMR and CD spectroscopy indicate this small fusion module comprises a left-handed polyproline type II (PPII) helix flanked by small, unstructured N and C termini. Individual prolines in the 6-residue proline-rich motif are highly tolerant of alanine substitutions, but multiple substitutions that disrupt the PPII helix eliminate cell-cell fusion activity. A synthetic p15 ectodomain peptide induces lipid mixing between liposomes, but with unusual kinetics that involve a long lag phase before the onset of rapid lipid mixing, and the length of the lag phase correlates with the kinetics of peptide-induced liposome aggregation. Lipid mixing, liposome aggregation, and stable peptide-membrane interactions are all dependent on both the N-terminal myristate and the presence of the PPII helix. We present a model for the mechanism of action of this novel viral fusion peptide, whereby the N-terminal myristate mediates initial, reversible peptide-membrane binding that is stabilized by subsequent amino acid-membrane interactions. These interactions induce a biphasic membrane fusion reaction, with peptide-induced liposome aggregation representing a distinct, rate-limiting event that precedes membrane merger. Although the prolines in the proline-rich motif do not directly interact with membranes, the PPII helix may function to force solvent exposure of hydrophobic amino acid side chains in the regions flanking the helix to promote membrane binding, apposition, and fusion.

Understanding the structural ensembles of a highly extended disordered protein

Developing a comprehensive description of the equilibrium structural ensembles for intrinsically disordered proteins (IDPs) is essential to understanding their function. The p53 transactivation domain (p53TAD) is an IDP that interacts with multiple protein partners and contains numerous phosphorylation sites. Multiple techniques were used to investigate the equilibrium structural ensemble of p53TAD in its native and chemically unfolded states. The results from these experiments show that the native state of p53TAD has dimensions similar to a classical random coil while the chemically unfolded state is more extended. To investigate the molecular properties responsible for this behavior, a novel algorithm that generates diverse and unbiased structural ensembles of IDPs was developed. This algorithm was used to generate a large pool of plausible p53TAD structures that were reweighted to identify a subset of structures with the best fit to small angle X-ray scattering data. High weight structures in the native state ensemble show features that are localized to protein binding sites and regions with high proline content. The features localized to the protein binding sites are mostly eliminated in the chemically unfolded ensemble; while, the regions with high proline content remain relatively unaffected. Data from NMR experiments support these results, showing that residues from the protein binding sites experience larger environmental changes upon unfolding by urea than regions with high proline content. This behavior is consistent with the urea-induced exposure of nonpolar and aromatic side-chains in the protein binding sites that are partially excluded from solvent in the native state ensemble.

Antibiotic Selection by the Promiscuous Aminoglycoside Acetyltransferase-(3)-IIIb Is Thermodynamically Achieved through the Control of Solvent Rearrangement

The results presented here show the first known observation of opposite signs of change in heat capacity (ΔC(p)) of two structurally similar ligands binding to the same protein site. Neomycin and paromomycin are aminoglycoside antibiotics that are substrates for the resistance-conferring enzyme, the aminoglycoside acetyltransferase-(3)-IIIb (AAC). These antibiotics are identical to one another except at the 6' position where neomycin has an amine and paromomycin has a hydroxyl. The opposite trends in ΔC(p) of binding of these two drugs to AAC suggest a differential exposure of nonpolar amino acid side chains. Nuclear magnetic resonance experiments further demonstrate significantly different changes in AAC upon interaction with neomycin and paromomycin. Experiments in H(2)O and D(2)O reveal the first observed temperature dependence of solvent and vibrational contributions to ΔC(p). Coenzyme A significantly influences these effects. Together, the data suggest that AAC exploits solvent properties to facilitate favorable thermodynamic selection of antibiotics.