Hydrophobic Surface Area Research Articles

The Effect of Parkinson's-Disease-Associated Mutations on the Deubiquitinating Enzyme UCH-L1

The neuronal ubiquitin C-terminal hydrolase (UCH) UCH-L1 has been linked to Parkinson's disease (PD) and other neurodegenerative diseases. Here, we present a study on the structure, stability, unfolding, and dynamics of wild-type and mutant UCH-L1. Fluorescence, far-UV CD, and NMR measurements were used to establish that the unfolding of UCH-L1 is three-state under equilibrium conditions and that an intermediate is populated. S18Y and I93M mutants, which are associated with a decreased risk or an increased risk of PD, respectively, are less stable than wild type. However, while there is minimal structural perturbation in the S18Y mutant, the I93M mutation is more disruptive. In particular, the NMR data suggest that there are local rearrangements around the site of the mutation, which we propose results in the exposure of hydrophobic surface area. This may have two consequences: an increased tendency towards, firstly, aggregation in vivo, and, secondly, aberrant interactions with tubulin and the chaperone-mediated autophagy machinery as observed by other groups, both of which may be involved in neurodegenerative processes.

一義集合体を形成するための疎水表面エンジニアリング

Exactly defined number of gear-shaped amphiphiles self-assemble into discrete aggregates. In water (or aqueous methanol) these amphiphiles mesh each other so as to reduce indented hydrophobic surface area exposed to the solvent, leading to 2 nm-sized cubic-shaped hexamers. Furthermore, tetrahedron-shaped tetramers are constructed from four gear-shaped amphiphiles and a guest molecule with the aid of van der Waals interaction between the amphiphiles and the guest molecule.

Identification of Interacting Hot Spots in the β3 Integrin Stalk Using Comprehensive Interface Design

Protein-protein interfaces are usually large and complementary surfaces, but specific side chains, representing energetic "hot spots," often contribute disproportionately to binding free energy. We used a computational method, comprehensive interface design, to identify hot spots in the interface between the stalk regions of the β3 and the complementary αIIb and αv integrin subunits. Using the Rosetta alanine-scanning and design algorithms to predict destabilizing, stabilizing, and neutral mutations in the β3 region extending from residues Lys(532) through Gly(690), we predicted eight alanine mutations that would destabilize the αIIbβ3 interface as well as nine predicted to destabilize the αvβ3 interface, by at least 0.3 kcal/mol. The mutations were widely and unevenly distributed, with four between residues 552 and 563 and five between 590 and 610, but none between 565 and 589, and 611 and 655. Further, mutations destabilizing the αvβ3 and αIIbβ3 interfaces were not identical. The predictions were then tested by introducing selected mutations into the full-length integrins expressed in Chinese hamster ovary cells. Five mutations predicted to destabilize αIIb and β3 caused fibrinogen binding to αIIbβ3, whereas three of four predicted to be neutral or stabilizing did not. Conversely, a mutation predicted to destabilize αvβ3, but not αIIbβ3 (D552A), caused osteopontin binding to αvβ3, but not fibrinogen binding to αIIbβ3. These results indicate that stability of the distal stalk interface is involved in constraining integrins in stable, inactive conformations. Further, they demonstrate the ability of comprehensive interface design to identify functionally significant integrin mutations.

Hydroxypropyl-Substituted β-Cyclodextrins: Influence of Degree of Substitution on the Thermodynamics of Complexation with Tauroconjugated and Glycoconjugated Bile Salts

The effect of the degree of substitution (DS) on the ability of hydroxypropylated β-cyclodextrin (HPβCD) to form inclusion complexes with six different bile salts, found within the intestinal tracts of rats, dogs, and humans, was studied by isothermal titration calorimetry. The composition and molecular structure of the cyclodextrin samples were characterized by MALDI-TOF mass spectrometry together with 1D and 2D-NMR, and some of the complexes were studied by 2D ROESY NMR. The stability and structure of the complexes were mainly determined by the position of hydroxyl groups on the bile salts and depended relatively little on the number of hydroxypropyl side chains on the CDs. The enthalpy and entropy of complexation exhibited a strong linear increase as the DS increased from 0 to 1, and a pronounced enthalpy-entropy compensation was observed. These observations are interpreted as an increased release of ordered water from the hydration shells of the bile salts, caused by the hydroxypropyl substituents on the rim of the CD. It is estimated that each CD hydroxypropyl substituent dehydrates a hydrophobic surface area of approximately 10 Å(2).

Molecular Dynamics Simulations of a β‐Hairpin Fragment of Protein G by Means of Atom‐Bond Electronegativity Equalization Method Fused into Molecular Mechanics (ABEEMδπ/MM)

AbstractThere are some controversial opinions about the origin of folding β‐hairpin stability in aqueous solution. In this study, the structural and dynamic behavior of a 16‐residue β‐hairpin from B1 domain of protein G has been investigated at 280, 300, 350 and 450 K using molecular dynamics (MD) simulations by means of Atom‐Bond Electronegativity Equalization Method Fused into Molecular Mechanics i.e., ABEEMδπ/MM and the explicit ABEEM‐7P water solvent model. In addition, a 300 K simulation of one mutant having the aromatic residues substituted with alanines has been performed. The hydrophobic surface area, hydrophilic surface area and some structural properties have been used to measure the role of the hydrophobic interactions. It is found that the aromatic residues substituted with alanines have shown an evident destabilization of the structure and unfolding started after 1.5 ns. It is also found that the number of the main chain hydrogen bonds have different distributions through three different simulations. All above demonstrate that the hydrophobic interactions and the main chain hydrogen bonds play an important role in the stability of the folding structure of β‐hairpin in solution. Furthermore, through the structural analyses of the β‐hairpin structures from four temperature simulations and the comparison with other MD simulations of β‐hairpin peptides, the new ABEEMδπ force field can reproduce the structural data in good agreement with the experimental data.

QSAR model for the prediction of bio-concentration factor using aqueous solubility and descriptors considering various electronic effects

The in silico modelling of bio-concentration factor (BCF) is of considerable interest in environmental sciences, because it is an accepted indicator for the accumulation potential of chemicals in organisms. Numerous QSAR models have been developed for the BCF, and the majority utilize the octanol/water partition coefficient (log P) to account for the penetration characteristics of the chemicals. The present work used descriptors from a variety of software packages for the development of a multi-linear regression model to estimate BCF. The modelled data set of 473 diverse compounds covers a wide range of log BCF values. In the proposed QSAR model, most of the variation is described by the calculated solubility in water. Other contributing descriptors describe, for instance, hydrophobic surface area, hydrogen bonding and other electronic effects. The model was validated internally by using a variety of statistical approaches. Two external validations were also performed. For the former validation, a subset from the same data source was used. The 2nd external validation was based on an independent data set collected from different resources. All validations showed the consistency of the model. The applicability domain of the model was discussed and described and a thorough outlier analysis was performed.

Importance of Surface Patterns for Defect Mitigation in Three-Dimensional Self-Assembly

This article investigates the three-dimensional self-assembly of submillimeter scale polyhedra using surface forces. Using a combination of energy landscape calculations and experiments, we investigate the influence of patterns of hydrophobic surfaces on generating defect-free, closed-packed aggregates of polyhedra, with a focus on cubic units. Calculations show that surface patterning strongly affects the interaction between individual units as well as that of the unit with the growing assembly. As expected, an increase in the hydrophobic surface area on each face results in larger global minima. However, it is the distribution of hydrophobic surface area on each cubic face that is strongly correlated to the energetic parameters driving low-defect assembly. For patterns with the same overall area, minimizing the radius of gyration and maximizing the angular distribution leads to steep energy curves, with a lower propensity for entrapment in metastable states. Experimentally, 200-500 microm sized metallic polyhedra were fabricated using a self-folding process, and the exposed surfaces were coated with a hydrophobic polymer. Cubes with surface patterns were agitated to cause aggregative self-assembly. Experimental results were consistent with energy calculations and suggest that geometric patterns with large overall areas, low radii of gyration, and high angular distributions result in efficient and low-defect assembly.

Protein Core Packing by Dynamic Combinatorial Chemistry

We describe the first example of the recursive selection of biologically relevant macromolecules from a dynamic combinatorial library (DCL). A small library of 36 peptides was allowed to undergo self-association in aqueous solution to form 8436 trimers. The stability of each of these trimers was governed by the formation of a well-packed hydrophobic core. The DCL allowed variation of the hydrophobic residues comprising this core over all combinations of glycine, alanine, valine, leucine, isoleucine, and phenylalanine at six positions. The study leads to three important conclusions: (i) fewer than 0.2% of all possible core packing arrangements have high folding stabilities; (ii) these arrangements are stabilized by intimate "jigsaw" packing, not by sequestration of maximum hydrophobic surface area; (iii) a well-defined "rule" for packing of stable cores exists, but this rule is nuanced by the presence of two unexpected amino acid sequences and the absence of one expected amino acid sequence.

RELATIONSHIP BETWEEN THE CALCULATED PHYSICOCHEMICAL PARAMETERS AND REVERSED PHASE THIN-LAYER CHROMATOGRAPHIC RETENTION BEHAVIOR OF CARBOXAMIDE FUNGICIDES AND RELATED COMPOUNDS

The lipophilicity of 6 carboxamide fungicides and 11 phenylbenzamide derivatives were determined by reversed phase layer chromatography (RP-TLC) employing silica and alumina as stationary phases impregnated with paraffin oil. Mixtures of methanol-water, acetonitrile-water, tetrahydrofurane-water, and acetone-water were used as mobile phases. The RM0 and b values related to the molecular lipophilicity and to the specific hydrophobic surface area (b) of the solutes were calculated separately for each RP-TLC system and for each analyte. The correlations between the physicochemical parameters measured were calculated by linear regression analysis. The impact of the site and character of the substituent on the retention of analytes was assessed by Free-Wilson analysis. The measured lipophilicity parameters were compared with 24 molecular descriptors computed applying free engines of the web. Calculations proved that the character of the stationary phase and the type of organic modifier exerted a negligible effect on the measured lipophilicity parameters. In the majority of cases the RM0 and b values and computed molecular descriptors were intercorrelated, but the fitness of the points to the linear regression equation was not strong enough to replace the measured values with the computed ones. This finding indicates the importance of using not only computed but also measured parameters for the future studies of both quantitative structure retention relationship (QSRR) and quantitative structure activity relationship (QSAR) calculations.

Modulating Native-like Residual Structure in the Fully Denatured State of Photoactive Yellow Protein Affects Its Refolding

Residual structure in the fully unfolded state is a key element for understanding protein folding. We show that the residual structure in fully denatured photoactive yellow protein (PYP) is affected by isomerization of its p-coumaric acid (pCA) chromophore. The exposure of total surface area and hydrophobic surface area upon unfolding was quantified by denaturant m values and heat capacity changes (DeltaC(p)), respectively. The exposure of the buried surface area upon the unfolding of the acid-denatured state of PYP containing trans-pCA is approximately 20% smaller than that of the native state. In contrast, for the partially unfolded pB photocycle intermediate containing cis-pCA, unfolding-induced exposure of the surface area is not decreased. These results show that pCA photoisomerization reduces residual structure in the fully unfolded state. Thus, residual structure in the fully unfolded state of PYP is under direct experimental control by photoexcitation. The sensitivity of the unfolded state to pCA isomerization provides a novel criterion that residual structure in the unfolded state of PYP is native-like, involving native-like protein-chromophore interactions. A largely untested prediction is that native-like residual structure facilitates the conformational search during folding. In the case of PYP, refolding from the less disordered fully unfolded state containing trans-pCA indeed is substantially accelerated. The burial of hydrophobic surface area in the fully unfolded state suggests that a significant part of the hydrophobic collapse process already has occurred in the denatured state.

Equilibrium and kinetics of the unfolding and refolding of Escherichia coli Malate Synthase G monitored by circular dichroism and fluorescence spectroscopy

The equilibrium and kinetics studies of an 82 kDa large monomeric Escherichia coli protein Malate Synthase G (MSG) was investigated by far and near-UV CD, intrinsic tryptophan fluorescence and extrinsic fluorescence spectroscopy. We find that despite of its large size, folding is reversible, in vitro. Equilibrium unfolding process of MSG exhibited three-state transition thus, indicating the presence of at least a stable equilibrium intermediate. Thermodynamic parameters suggest this intermediate resembles the unfolded state. However, the equilibrium intermediate exhibits pronounced secondary structure as measured by far-UV CD, partial tertiary structure as delineated by near-UV CD, compactness (m value) and exposed hydrophobic surface area as assessed by ANS binding, typically depicting a molten globule state. The stopped-flow kinetic data provide clear evidence for the presence of a burst phase during the refolding pathway due to the formation of an early Intermediate, within the dead time of the instrument. Refolding from 4 M to various lower concentrations until 0.4 M of GdnHCl follow biphasic kinetics at lower concentrations of GdnHCl (<0.8 M), whereas monophasic kinetics at concentrations above 1.5 M. Also, rollover in the refolding and unfolding limbs of chevron plot verifies the presence of a fast kinetic intermediate at lower concentration of GdnHCl. Based upon the above observations we hereby propose the folding pathway of a large multi-domain protein Malate Synthase G.

Thermodynamic Analysis of a Molecular Chaperone Binding to Unfolded Protein Substrates

Molecular chaperones are a highly diverse group of proteins that recognize and bind unfolded proteins to facilitate protein folding and prevent nonspecific protein aggregation. The mechanisms by which chaperones bind their protein substrates have been studied for decades. However, there are few reports about the affinity of molecular chaperones for their unfolded protein substrates. Thus, little is known about the relative binding affinities of different chaperones and about the relative binding affinities of chaperones for different unfolded protein substrates. Here we describe the application of SUPREX (stability of unpurified proteins from rates of H-D exchange), an H-D exchange and MALDI-based technique, in studying the binding interaction between the molecular chaperone Hsp33 and four different unfolded protein substrates, including citrate synthase, lactate dehydrogenase, malate dehydrogenase, and aldolase. The results of our studies suggest that the cooperativity of the Hsp33 folding-unfolding reaction increases upon binding with denatured protein substrates. This is consistent with the burial of significant hydrophobic surface area in Hsp33 when it interacts with its substrate proteins. The SUPREX-derived K(d) values for Hsp33 complexes with four different substrates were all found to be within the range of 3-300 nM.

Acid hybrid catalysts from poly(styrenesulfonic acid) grafted onto ultra-large-pore SBA-15 silica using atom transfer radical polymerization

Poly(styrenesulfonic acid)-functionalized materials based on poly(styrenesulfonic acid sodium salt) incorporated via aqueous atom transfer radical polymerization (ATRP) initiated from the surface of large-pore mesoporous SBA-15 silica support have been synthesized. The inorganic–organic nature of these hybrid materials makes them particularly desirable for acid-catalyzed reactions that require extended and hydrophobic surface areas with a narrow pore diameter distribution in the mesoporous range. Acidic hybrid materials were prepared by grafting the ATRP-initiator (3-(chlorodimethylsilyl)propyl bromoisobutyrate) on the silica surface, subsequent polymerization of the styrenesulfonic acid sodium salt monomer, and final sodium ion exchange by acid activation. Conventional and ultra-large-pore SBA-15 silica supports with nominal (BJH) pore diameter ranging from 8 to 32 nm were used for the incorporation of different polymer loadings at different polymerization times. The silylation of ATRP-initiator-functionalized SBA-15 supports has allowed a better control of the ATRP within the mesoporous structure. The use of ultra-large-pore SBA-15 supports provides a remarkable increase of the porosity which allowed us to properly allocate the polymer. The hybrid poly(styrenesulfonic acid)-modified materials showed good catalytic activities in the esterification of oleic acid with n-butanol, particularly in terms of intrinsic activity per acid site.

Abstract A141: Synthesis and bioactivity of core-expanded calixarene-based antitumor agents

Abstract Anginex, a synthetic 33mer β-sheet-forming peptide, is a potent inhibitor of angiogenesis in vitro and in vivo. Previous studies demonstrate that Anginex, through binding to one of the hydrophobic faces of human galectin-1 (hGal-1), prevents endothelial cell adhesion to the extracellular matrix, induces apoptosis in angiogenically-activated endothelial cells (ECs), and suppresses tumor growth in animal models. Since small molecules have several pharmacologic advantages over therapeutic peptides, we sought to design a small molecule peptidomimetic of anginex. Calix[4]arene was chosen as the basic scaffold to mimic the approximate molecular dimensions, amphipathicity, and cationic topology of Anginex. Chemical modification of the hydrophobic (upper rim) and hydrophilic (lower rim) faces of calix[4]arene resulted in the discovery of a lead compound, 0118, possessing in vitro and in vivo activities similar to that of anginex. These studies reveal that small hydrophobic groups on the upper rim and dimethylaminoethylacetamide (DMAEA) moieties on the lower rim of 0118 are important for its biologic activity. Until now, structure-activity relationship (SAR) development of calixarene has focused on modifications of its hydrophobic and hydrophilic surfaces. Since the hGal-1-Anginex binding site is a hydrophobic surface, we hypothesize that a larger hydrophobic calixarene surface area will translate into enhanced bioactivity. We have chosen calix[6]arene and tetrathiacalix[4]arene as tools to explore the core-expanded SAR of our lead compound. A calix[6]arene analog of 0118 not only has a larger hydrophobic footprint, but also a greater intra-annular topoisomerization rate which should allow for greater conformational space sampling and, therefore, better inducible fit within its molecular receptor. The tetrathiacalix[4]arene derivative, with its longer C-S bonds (relative to the methylene bonds of calix[4]arene and calix[6]arene), should allow for a hydrophobic footprint larger than the parent 0118 but smaller than that of the calix[6]arene analog. The current studies focus on the synthesis and biologic activity of these 0118 core-expanded derivatives. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A141.

Intrinsic Structural Disorder Confers Cellular Viability on Oncogenic Fusion Proteins

Chromosomal translocations, which often generate chimeric proteins by fusing segments of two distinct genes, represent the single major genetic aberration leading to cancer. We suggest that the unifying theme of these events is a high level of intrinsic structural disorder, enabling fusion proteins to evade cellular surveillance mechanisms that eliminate misfolded proteins. Predictions in 406 translocation-related human proteins show that they are significantly enriched in disorder (43.3% vs. 20.7% in all human proteins), they have fewer Pfam domains, and their translocation breakpoints tend to avoid domain splitting. The vicinity of the breakpoint is significantly more disordered than the rest of these already highly disordered fusion proteins. In the unlikely event of domain splitting in fusion it usually spares much of the domain or splits at locations where the newly exposed hydrophobic surface area approximates that of an intact domain. The mechanisms of action of fusion proteins suggest that in most cases their structural disorder is also essential to the acquired oncogenic function, enabling the long-range structural communication of remote binding and/or catalytic elements. In this respect, there are three major mechanisms that contribute to generating an oncogenic signal: (i) a phosphorylation site and a tyrosine-kinase domain are fused, and structural disorder of the intervening region enables intramolecular phosphorylation (e.g., BCR-ABL); (ii) a dimerisation domain fuses with a tyrosine kinase domain and disorder enables the two subunits within the homodimer to engage in permanent intermolecular phosphorylations (e.g., TFG-ALK); (iii) the fusion of a DNA-binding element to a transactivator domain results in an aberrant transcription factor that causes severe misregulation of transcription (e.g. EWS-ATF). Our findings also suggest novel strategies of intervention against the ensuing neoplastic transformations.

Zeolite-Templated Microporous Carbon As a Superior Adsorbent for Removal of Monoaromatic Compounds from Aqueous Solution

A microporous carbon with very high specific surface area and narrow pore size distribution was synthesized using Y zeolite as a template. The structural, porosity, and surface characteristics of the material were investigated by elemental analysis, N2 adsorption, powder X-ray diffraction, and Raman spectroscopy. The batch adsorption technique was performed to assess adsorption of three monoaromatic compounds, phenol, 1,3-dichlorobenzene, and 1,3-dinitrobenzene, on the synthesized carbon. Nonporous graphite, single-walled carbon nanotubes, and two commercial microporous activated carbons were also included as comparative adsorbents. The synthesized microporous carbon showed extraordinarily high adsorption affinity (comparable or higher than activated carbons and carbon nanotubes) for the three adsorbates, and very fast adsorption/ desorption kinetics (equilibrium reached less than 3 h) and complete adsorption reversibility for phenol. These adsorption properties were attributed to the large hydrophobic surface area and the regular-shaped, open and interconnected three-dimensional pore structure of the synthesized microporous carbon. Additionally, with normalization of adsorbent surface area adsorption of a bulky solute, 1,2,4,5-tetrachlorobenzene, was prominently higher on the synthesized carbon than on the activated carbons, due to alleviated size exclusion effect. Findings of the present work highlight the potential of using zeolite-templated carbons as effective adsorbents for removal of hydrophobic organic contaminants in water treatment.

Phosphorylation of hormone‐sensitive lipase by protein kinase A in vitro promotes an increase in its hydrophobic surface area

Hormone-sensitive lipase (EC 3.1.1.79; HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. HSL activity is controlled by phosphorylation of at least four serines. In rat HSL, Ser563, Ser659 and Ser660 are phosphorylated by protein kinase A (PKA) in vitro as well as in vivo, and Ser660 and Ser659 have been shown to be the activity-controlling sites in vitro. The exact molecular events of PKA-mediated activation of HSL in vitro are yet to be determined, but increases in both Vmax and S0.5 seem to be involved, as recently shown for human HSL. In this study, the hydrophobic fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) was found to inhibit the hydrolysis of triolein by purified recombinant rat adipocyte HSL, with a decrease in the effect of bis-ANS upon PKA phosphorylation of HSL. The interaction of HSL with bis-ANS was found to have a Kd of 1 microM in binding assays. Upon PKA phosphorylation, the interactions of HSL with both bis-ANS and the alternative probe SYPRO Orange were increased. By negative stain transmission electron microscopy, phosphorylated HSL was found to have a closer interaction with phospholipid vesicles than unphosphorylated HSL. Taken together, our results show that HSL increases its hydrophobic nature upon phosphorylation by PKA. This suggests that PKA phosphorylation induces a conformational change that increases the exposed hydrophobic surface and thereby facilitates binding of HSL to the lipid substrate.

Amino Acid Insertion Reveals a Necessary Three-Helical Intermediate in the Folding Pathway of the Colicin E7 Immunity Protein Im7

The small (87-residue) α-helical protein Im7 (an inhibitor protein for colicin E7 that provides immunity to cells producing colicin E7) folds via a three-state mechanism involving an on-pathway intermediate. This kinetic intermediate contains three of four native helices that are oriented in a non-native manner so as to minimise exposed hydrophobic surface area at this point in folding. The short (6-residue) helix III has been shown to be unstructured in the intermediate ensemble and does not dock onto the developing hydrophobic core until after the rate-limiting transition state has been traversed. After helix III has docked, it adopts an α-helical secondary structure, and the side chains of residues within this region provide contacts that are crucial to native-state stability. In order to probe further the role of helix III in the folding mechanism of Im7, we created a variant that contains an eight-amino-acid polyalanine-like helix stabilised by a Glu-Arg salt bridge and an Asn-Pro-Gly capping motif, juxtaposed C-terminal to the natural 6-residue helix III. The effect of this insertion on the structure of the native protein and its folding mechanism were studied using NMR and ϕ-value analysis, respectively. The results reveal a robust native structure that is not perturbed by the presence of the extended helix III. Mutational analysis performed to probe the folding mechanism of the redesigned protein revealed a conserved mechanism involving the canonical three-helical intermediate. The results suggest that folding via a three-helical species stabilised by both native and non-native interactions is an essential feature of Im7 folding, independent of the helical propensity of helix III.

Importance of non-conserved distal carboxyl terminal amino acids in two peptidases belonging to the M1 family: Thermoplasma acidophilum Tricorn interacting factor F2 and Escherichia coli Peptidase N

Enzymes belonging to the M1 family play important cellular roles and the key amino acids (aa) in the catalytic domain are conserved. However, C-terminal domain aa are highly variable and demonstrate distinct differences in organization. To address a functional role for the C-terminal domain, progressive deletions were generated in Tricorn interacting factor F2 from Thermoplasma acidophilum (F2) and Peptidase N from Escherichia coli (PepN). Catalytic activity was partially reduced in PepN lacking 4 C-terminal residues (PepNΔC4) whereas it was greatly reduced in F2 lacking 10 C-terminal residues (F2ΔC10) or PepN lacking eleven C-terminal residues (PepNΔC11). Notably, expression of PepNΔC4, but not PepNΔC11, in E. coliΔ pepN increased its ability to resist nutritional and high temperature stress, demonstrating physiological significance. Purified C-terminal deleted proteins demonstrated greater sensitivity to trypsin and bound stronger to 8-amino 1-napthalene sulphonic acid (ANS), revealing greater numbers of surface exposed hydrophobic aa. Also, F2 or PepN containing large aa deletions in the C-termini, but not smaller deletions, were present in high amounts in the insoluble fraction of cell extracts probably due to reduced protein solubility. Modeling studies, using the crystal structure of E. coli PepN, demonstrated increase in hydrophobic surface area and change in accessibility of several aa from buried to exposed upon deletion of C-terminal aa. Together, these studies revealed that non-conserved distal C-terminal aa repress the surface exposure of apolar aa, enhance protein solubility, and catalytic activity in two soluble and distinct members of the M1 family.

Dimer Formation of a Stabilized Gβ1 Variant: A Structural and Energetic Analysis

In previous work, a strongly stabilized variant of the β1 domain of streptococcal protein G (Gβ1) was obtained by an in vitro selection method. This variant, termed Gβ1-M2, contains the four substitutions E15V, T16L, T18I, and N37L. Here we elucidated the molecular basis of the observed strong stabilizations. The contributions of these four residues were analyzed individually and in various combinations, additional selections with focused Gβ1 gene libraries were performed, and the crystal structure of Gβ1-M2 was determined. All single substitutions (E15V, T16L, T18I, and N37L) stabilize wild-type Gβ1 by contributions of between 1.6 and 6.0 kJ mol − 1 (at 70 °C). Hydrophobic residues at positions 16 and 37 provide the major contribution to stabilization by enlarging the hydrophobic core of Gβ1. They also increase the tendency to form dimers, as shown by dependence on the concentration of apparent molecular mass in analytical ultracentrifugation, by concentration-dependent stability, and by a strongly increased van't Hoff enthalpy of unfolding. The 0.88-Å crystal structure of Gβ1-M2 and NMR measurements in solution provide the explanation for the observed dimer formation. It involves a head-to-head arrangement of two Gβ1-M2 molecules via six intermolecular hydrogen bonds between the two β strands 2 and 2′ and an adjacent self-complementary hydrophobic surface area, which is created by the T16L and N37L substitutions and a large 120° rotation of the Tyr33 side chain. This removal of hydrophilic groups and the malleability of the created hydrophobic surface provide the basis for the dimer formation of stabilized Gβ1 variants.