bis-ANS Binding Research Articles

Structural and thermodynamic characterization of a highly amyloidogenic dimer of transthyretin involved in a severe cardiomyopathy

Transthyretin (TTR) is a homotetrameric protein involved in the transport of thyroxine. More than 150 different mutations have been described in the TTR gene, several of them associated with familial amyloid cardiomyopathy (FAC). Recently, our group described a new variant of TTR in Brazil, namely A39D-TTR, which causes a severe cardiac condition. Position 39 is in the AB loop, a region of the protein that is located within the thyroxine-binding channels and is involved in tetramer formation. In the present study we solved the structure and characterize the thermodynamic stability of this new variant of TTR using urea and high hydrostatic pressure (HHP). Interestingly, during the process of purification, A39D-TTR turned out to be a dimer and not a tetramer, a variation that might be explained by the close contact of the four aspartic acids at position 39, where they face each other inside the thyroxine channel. In the presence of sub-denaturing concentrations of urea, bis-ANS binding and dynamic light scattering revealed A39D-TTR in the form of a molten-globule dimer. Co-expression of A39D and WT isoforms in the same bacterial cell did not produce heterodimers or heterotetramers, suggesting that somehow a negative charge at the AB loop precludes tetramer formation. A39D-TTR proved to be highly amyloidogenic, even at mildly acidic pH values where WT-TTR does not aggregate. Interestingly, despite being a dimer, aggregation of A39D-TTR was inhibited by diclofenac, which binds to the thyroxine channel in the tetramer, suggesting the existence of other pockets in A39D-TTR able to accommodate this molecule.

The Role of Protein-L-isoaspartyl Methyltransferase (PIMT) in the Suppression of Toxicity of the Oligomeric Form of Aβ42, in Addition to the Inhibition of Its Fibrillization.

The oligomeric form of amyloid-β peptide (Aβ42) plays a crucial role in the pathogenesis of Alzheimer's disease (AD) and is responsible for cognitive deficits. The soluble oligomers are believed to be more toxic compared to the fibril form. Protein-L-isoaspartyl methyltransferase (PIMT) is a repair enzyme that converts aberrant isoAsp residues, formed spontaneously on isomerization of normal Asp and Asn residues, back to typical Asp. It was shown to inhibit the fibrillization of Aβ42 (containing three Asp residues), and here, we investigate its effect on the size, conformation, and toxicity of Aβ42 oligomers (AβO). Far-UV CD indicated a shift in the conformational feature of AβOs from the random coil to β-sheet in the presence of PIMT. Binding of bis-ANS to different AβOs (obtained using different concentrations of Aβ42 monomer) indicated the correlation of size of oligomers to hydrophobicity: the smallest AβO having the highest hydrophobicity is the most toxic. Dynamic light scattering showed an increase in size of AβO with the addition of PIMT, a contrasting role to that on Aβ fibril. Assays using PC12-derived neurons showed the neuroprotective role of PIMT against AβO-induced toxicity. Furthermore, we have elaborated on the molecular mechanism of the antifibrillar action of PIMT and how this function is correlated with its enzymatic activity. PIMT has a more pronounced effect on AβO as compared to a small heat shock protein, pointing to its importance for the amelioration of the adverse effect of both Aβ42 oligomers and fibrils.

Development of β-sheet structure in Aβ aggregation intermediates diminishes exposed hydrophobic surface area and enhances proinflammatory activity

Three decades of research, both in vitro and in vivo, have demonstrated the conformational heterogeneity that is displayed by the amyloid β peptide (Aβ) in Alzheimer's disease (AD). Understanding the distinct properties between Aβ conformations and how conformation may impact cellular activity remain open questions, yet still continue to provide new insights into protein misfolding and aggregation. In particular, there is interest in the group of soluble oligomeric prefibrillar Aβ species comprising lower molecular weight oligomers up to larger protofibrils. In the current study, a number of strategies were utilized to separate Aβ protofibrils and oligomers and show that the smaller Aβ oligomers have a much different conformation than Aβ protofibrils. The differences were consistent for both Aβ40 and Aβ42. Protofibrils bound thioflavin T to a greater extent than oligomers, and were highly enriched in β-sheet secondary structure. Aβ oligomers possessed a more open structure with significant solvent exposure of hydrophobic domains as determined by tryptophan fluorescence and bis-ANS binding, respectively. The protofibril-selective antibody AbSL readily discerned conformational differences between protofibrils and oligomers. The more developed structure for Aβ protofibrils ultimately proved critical for provoking the release of tumor necrosis factor α from microglial cells. The findings demonstrated a dependency on β-sheet structure for soluble Aβ aggregates to cause a microglial inflammatory response. The Aβ aggregation process yields many conformationally-varied species with different levels of β-structure and exposed hydrophobicity. The conformation elements likely determine biological activity and pathogenicity.

The N-terminal MYB domains affect the stability and folding aspects of Arabidopsis thaliana MYB4 transcription factor under thermal stress.

The MYB4 transcription factor, a member of R2R3-type subfamily of MYB domain protein, plays a key role in the regulation of accumulation of UV-B absorbing phenylpropanoids in Arabidopsis. Although UV-B and thermal stress generate some common stress response, the effect of elevated temperature on the conformational stability of MYB4 remains limited. This study describes the folding and aggregation properties of Arabidopsis MYB4 protein under thermal stress condition. Circular dichroism spectral studies and Bis-ANS binding assays have indicated that the removal of the N-terminal MYB domain affects the structural conformation of the protein and disrupts surface hydrophobic binding sites at higher temperature. Urea-induced equilibrium unfolding studies revealed that the removal of the N-terminal region lowers the thermodynamic stability of MYB4 at elevated temperature. Tryptophan fluorescence spectral pattern and both in vitro and in vivo aggregation studies have revealed the importance of the N-terminal second MYB domain encompassing the N-terminal 62-116 amino acid residues in regulating MYB4 protein stability at higher temperature. On the other hand, comparison of the growth response of wild-type Arabidopsis and atmyb4 mutant line have suggested that MYB4 may not directly affect plant response under thermal stress condition and only marginal role of MYB4 in controlling thermomorphogenesis in Arabidopsis. Interestingly, immunoprecipitation studies have revealed that HSP90 specifically interacts with MYB4 in vivo at the endogenous level, indicating the possible role of HSP90 in governing the stability of MYB4 at elevated temperature in Arabidopsis.

Physicochemical and structural properties of lunasin revealed by spectroscopic, chromatographic and molecular dynamics approaches

Lunasin is a 43-amino acid peptide from seeds and grains with bioavailability in humans and potent chemotherapeutic action against several cancer cell lines. Here, we investigate new information about the physicochemical and structural properties of lunasin using circular dichroism (CD), fluorescence spectroscopy, electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS), size exclusion chromatography (SEC), molecular dynamics (MD), and bioinformatics. CD analysis and disorder prediction obtained by PONDR indicate that lunasin has a mostly unordered structure. Double wavelength [θ]222nm x [θ]200nm plot data suggests that lunasin is an intrinsically disordered peptide (IDP) in a pre-molten globule-like (PMG-like) state, while CD spectrum deconvolution and MD simulation indicate small β-strand content. The presence of residual structure was supported by loss of CD signal at 222 nm after treatment with urea and by increasing fluorescence emission upon bis-ANS binding. Lunasin also demonstrated stability to heating up to the temperature of 100 °C, as verified by CD. MD and CD analyses in the presence of TFE and MoRFpred prediction indicated the helix propensity of lunasin. ESI-IMS-MS data revealed that lunasin shows a propensity to form disulfide bonds at the conditions used. MD data also indicated that disulfide bond formation affects the adopted structure, showing a possible role of aspartyl-end in structure stabilization and compaction. In conclusion, our data support a characterization of lunasin as a peptide with an intrinsic disorder in a PMG-like state and reveal new aspects about its structural stability and plasticity, as well as the effects of disulfide bond formation and electrostatic attractions.

An insight into the folding and stability of Arabidopsis thaliana SOG1 transcription factor under salinity stress in vitro

The present study describes the biophysical characterization of Arabidopsis thaliana SOG1 (SUPPRESSOR OF GAMMA RESPONSE 1) protein, a NAC domain transcription factor which plays central role in DNA damage response pathway, under salinity stress in vitro. Tryptophan fluorescence studies using purified recombinant wild type (full length) AtSOG1 and its N-terminal or C-terminal deletion forms (AtSOG1ΔNAC and AtSOG1ΔCT respectively) have revealed high salinity induced conformational change due to removal of the N-terminal NAC domain. Bis-ANS binding assays indicate that removal of the N-terminal NAC domain increases the surface hydrophobic binding sites, while the C-terminal region of SOG1 also plays important role in regulating the surface hydrophobicity aspects following exposure to high salinity. Circular dichroism (CD) spectral studies have indicated that removal of the N-terminal NAC domain affects the structural conformation of the protein under high salt concentration. Urea-induced equilibrium unfolding studies revealed decreased stability of C-terminal region due to removal of the N-terminal NAC domain. In vitro aggregation studies have indicated higher propensity of aggregation of AtSOG1ΔNAC due to salt treatment. Overall, our results provide evidence for the importance of both N-terminal NAC domain and the C-terminal region in regulating the stability of SOG1 protein under salinity stress in vitro.

The cmc-value of a bolalipid with two phosphocholine headgroups and a C24 alkyl chain: Unusual binding properties of fluorescence probes to bolalipid aggregates

Bolalipids with a long alkyl chain and two phosphocholine polar groups self-assemble in water into two different types of aggregate structures, namely helical nanofibers at low temperature and two types of micellar aggregates at higher temperature. We tried to determine the critical aggregation concentration (cac) or critical micellar concentration (cmc) of the bolalipid tetracosane-1,24-bis(phosphocholine) (PC-C24-PC) by using different fluorescent probes. The use of pyrene or pyrene derivatives as fluorophores failed, whereas the probes 1,8-ANS and particularly bis-ANS gave consistent results. The structure of the bolalipid aggregates obviously hinders partitioning or binding of pyrene derivatives into the micellar interior, whereas 1,8-ANS and bis-ANS can bind to the surface of the aggregate structures. The observed large increase in fluorescence intensity of bis-ANS indicates that binding to the hydrophobic surface of the aggregates leads to a reduction of the dye mobility. However, binding of bis-ANS is relatively weak, so that the determination of a cac/cmc-value is difficult. Simulations of the intensity curves for PC-C24-PC lead to estimates of the cac/cmc-value of 0.3–1.0×10−6M, depending on the structure of the aggregates. Single molecule fluorescence correlation spectroscopy was used to determine the mobility of bis-ANS as a function of concentration of PC-C24-PC. The dye diffusion time and the molecular brightness are lower at low bolalipid concentration, when only free dye is present, and increase at higher concentration when bis-ANS is bound to the aggregates. The experimental cac/cmc-values are higher than those estimated, using an incremental method for the change in Gibbs free energy for micellization with n-alkyl-phosphocholines with only one polar group as a comparison. Apparently, for PC-C24-PC in micellar or fibrous aggregates, more CH2 groups are exposed to water than in a conventional micelle of an n-alkyl-phosphocholine.

Nano-cage-mediated refolding of insulin by PEG-PE micelle

Insulin aggregation has pronounced pharmaceutical implications and biological importance. Deposition of insulin aggregates is associated with type II diabetes and instability of pharmaceutical formulations. We present in this study the renaturation effect of PEG-PE micelle on dithiothreitol (DTT)-denatured insulin revealed by techniques including turbidity assay, circular dichroism (CD), thioflavinT (ThT) binding assay, bis-ANS binding assay, agarose gel electrophoresis and MALDI-TOF MS. The obtained results show that PEG-PE micelle having a hydrophilic nano-cage-like structure in which with a negative charge layer, can capture DTT-induced insulin A and B chains, and block their hydrophobic interaction, thereby preventing aggregation. The reduced insulin A and B chain in the nano-cage are capable of recognizing each other and form the native insulin with yields of ∼30% as measured by hypoglycemic activity analysis in mice. The observed insulin refolding assisted by PEG-PE micelle may be applicable to other proteins.

Understanding the Physical and Molecular Basis of Stability of Arabidopsis DNA Pol λ under UV-B and High NaCl Stress.

Here, we have investigated the physical and molecular basis of stability of Arabidopsis DNA Pol λ, the sole X family DNA polymerase member in plant genome, under UV-B and salinity stress in connection with the function of the N-terminal BRCT (breast cancer-associated C terminus) domain and Ser-Pro rich region in the regulation of the overall structure of this protein. Tryptophan fluorescence studies, fluorescence quenching and Bis-ANS binding experiments using purified recombinant full length Pol λ and its N-terminal deletion forms have revealed UV-B induced conformational change in BRCT domain deficient Pol λ. On the other hand, the highly conserved C-terminal catalytic core PolX domain maintained its tertiary folds under similar condition. Circular dichroism (CD) and fourier transform infrared (FT-IR) spectral studies have indicated appreciable change in the secondary structural elements in UV-B exposed BRCT domain deficient Pol λ. Increased thermodynamic stability of the C-terminal catalytic core domain suggested destabilizing effect of the N-terminal Ser-Pro rich region on the protein structure. Urea-induced equilibrium unfolding studies have revealed increased stability of Pol λ and its N-terminal deletion mutants at high NaCl concentration. In vivo aggregation studies using transient expression systems in Arabidopsis and tobacco indicated possible aggregation of Pol λ lacking the BRCT domain. Immunoprecipitation assays revealed interaction of Pol λ with the eukaryotic molecular chaperone HSP90, suggesting the possibility of regulation of Pol λ stability by HSP90 in plant cell. Overall, our results have provided one of the first comprehensive information on the biophysical characteristics of Pol λ and indicated the importance of both BRCT and Ser-Pro rich modules in regulating the stability of this protein under genotoxic stress in plants.

Effects of Cofactors on Conformation Transition of Random Peptides Consisting of a Reduced Amino Acid Alphabet.

This study aims to explore the structure characteristic of random polypeptides constructed by origin early amino acid alphabet, as well as the effects of cofactors on conformation transition of random peptides. DNA library R8-4 encoding VNM random peptides were constructed by small cassette strategy. Subsequently, a random polypeptide library was constructed using in vitro translation. Expression and purification of VNM random peptides were also performed by a conventional method of recombinant. CD spectrum analysis indicated that VNM random polypeptides have a secondary structure characteristic of protein, such as the content of α-helix is greater than 60%, random coil is about 20% β sheet, and β turn is less than 10%. CD spectrum changed with the addition of 10-40 µM ATP and NADP, but slightly changed by NAD; no influence was observed with MgSO4. Bis-ANS binding assay indicated that fluorescent intensity of bis-ANS was strengthened slightly by 10 VNM random peptides. Fluorescent intensity was strengthened fourfold by adding 10-40 µM ATP, NAD, and NADH, whereas the inducing effect of NADPH and MgSO4 were negligible. VNM random peptides have a classic secondary structure and hydrophobic domain in water solution. Moreover, conformation transition and hydrophobic domain could be induced by cofactor, indicating the preliminary evidence for the hypothesis that "the origin of primitive protein was induced by small molecule."

Quaternary structure of human small heat shock protein HSPB6 (Hsp20) in crowded media modeled by trimethylamine N-oxide (TMAO): Effect of protein phosphorylation.

Effect of trimethylamine N-oxide (TMAO), well-known osmolyte, widely used to imitate crowded intracellular conditions, on the quaternary structure of recombinant human small heat shock protein HspB6 (Hsp20) was analyzed by means of size-exclusion chromatography, chemical crosslinking and analytical ultracentrifugation. Consistent with previous reports, in the absence of TMAO unphosphorylated, pseudophosphorylated (S16D mutant) and phosphorylated HspB6 form only small oligomers (presumably dimers). Addition of TMAO to unphosphorylated HspB6 leads to formation of different large oligomers being in equilibrium with dimers. Pseudophosphorylation (S16D mutation) or phosphorylation partially or completely prevent TMAO-induced oligomerization of HspB6. Pseudophosphorylation affects bis-ANS binding suggesting decreased hydrophobicity of HspB6. According to size-exclusion chromatography, TMAO-induced changes of HspB6 oligomerization result in its altered interaction with HspB1 and this effect can be reversed by HspB6 phosphorylation. It is concluded that under conditions of molecular crowding, characteristic for intracellular environment, HspB6 undergoes reversible changes of its oligomeric state which can affect its physiologically important properties and can be delicately regulated by phosphorylation.

The p10 FAST protein fusion peptide functions as a cystine noose to induce cholesterol-dependent liposome fusion without liposome tubulation

The reovirus p10 fusion-associated small transmembrane (FAST) proteins are the smallest known membrane fusion proteins, and evolved specifically to mediate cell–cell, rather than virus–cell, membrane fusion. The 36–40-residue ectodomains of avian reovirus (ARV) and Nelson Bay reovirus (NBV) p10 contain an essential intramolecular disulfide bond required for both cell–cell fusion and lipid mixing between liposomes. To more clearly define the functional, biochemical and biophysical features of this novel fusion peptide, synthetic peptides representing the p10 ectodomains of ARV and NBV were analyzed by solution-state NMR spectroscopy, circular dichroism spectroscopy, fluorescence spectroscopy-based hydrophobicity analysis, and liposome binding and fusion assays. Results indicate that disulfide bond formation promotes exposure of hydrophobic residues, as indicated by bis-ANS binding and time-dependent peptide aggregation under aqueous conditions, implying the disulfide bond creates a small, geometrically constrained, cystine noose. Noose formation is required for peptide partitioning into liposome membranes and liposome lipid mixing, and electron microscopy revealed that liposome–liposome fusion occurs in the absence of liposome tubulation. In addition, p10 fusion peptide activity, but not membrane partitioning, is dependent on membrane cholesterol.

Doubling the cross-linking interface of a rationally designed beta roll peptide for calcium-dependent proteinaceous hydrogel formation.

We have rationally engineered a stimulus-responsive cross-linking domain based on a repeats-in-toxin (RTX) peptide to enable calcium-dependent formation of supramolecular hydrogel networks. The peptide isolated from the RTX domain is intrinsically disordered in the absence of calcium. In calcium rich environments, the peptide binds Ca(2+) ions and folds into a beta roll (β-roll) secondary structure composed to two parallel β-sheet faces. Previously, we mutated one of the faces to contain solvent exposed leucine side chains which are localized only in the calcium-bound β-roll conformation. We demonstrated the ability of this mutant peptide to self-assemble into hydrogels in the presence of calcium with the aid of additional peptide-based cross-linking moieties. Here, we have expanded this approach by engineering both β-roll faces to contain leucine residues, thereby doubling the cross-linking interface for each monomeric building block. These leucine rich surfaces impart a hydrophobic driving force for self-assembly. Extensive characterization was performed on this double-faced mutant to ensure the retention of calcium affinity and subsequent structural rearrangement similar to that of the wild type domain. We genetically fused an α-helical leucine zipper capable of forming tetrameric coiled-coil bundles to the peptide and the resulting chimeric protein self-assembles into a hydrogel only in calcium rich environments. Since this new mutant peptide enables cross-linking on both surfaces simultaneously, a higher oligomerization state was achieved. To further investigate the cross-linking capability, we constructed concatemers of the β-roll with maltose binding protein (MBP), a monomeric globular protein, without the leucine zipper domains. These concatemers show a similar sol-gel transition in response to calcium. Several biophysical techniques were used to probe the structural and mechanical properties of the mutant β-roll domain and the resulting supramolecular networks including circular dichroism, fluorescence resonance energy transfer, bis-ANS binding, and microrheology. These results demonstrate that the engineered β-roll peptides can mediate calcium-dependent cross-linking for protein hydrogel formation without the need for any other cross-linking moieties.

Binding of bis-ANS to Bacillus subtilis lipase: A combined computational and experimental investigation

8-Anilino-1-naphthalene sulfonate (ANS) and its covalent dimer bis-ANS are widely used for titrating hydrophobic surfaces of proteins. Interest to understand the nature of interaction of these dyes with proteins was seriously pursued. However as the techniques used in these studies varied, they often provided varied information regarding stoichiometry, binding affinity, actual binding sites etc. In the present study, we used combination of computation methods (docking and MD simulation) and experimental methods (mutations, steady-state and time-resolved fluorescence) to investigate bis-ANS interaction with Bacillus subtilis lipase. We identified seven binding sites for bis-ANS on lipase using computational docking and MD simulation and verified these data using a set of single amino acid substituted mutants. Docking and MD simulation studies indicated that the binding sites were various indentations and grooves on protein surface with hydrophobic characteristics. Both hydrophobic and ionic interactions were involved in each of these binding events. We further examine the fluorescence properties of bis-ANS bound to mutant lipases that either gained or lost a binding site. Our results indicated that neither gain nor loss of single binding site caused any change in fluorescence lifetimes (and their relative amplitudes) of mutant lipase-bound bis-ANS in comparison to that bound to wild type; hence, it suggested that nature of bis-ANS binding to each of the sites in lipase was very similar.

An analysis of the factors that affect the dissociation of inclusion bodies and the refolding of endostatin under high pressure

An optimization of the refolding of endostatin (ES), by a study of the conditions that can affect (i) dissociation of inclusion bodies (IBs) and (ii) renaturation under high hydrostatic pressure (HHP), is described. IBs produced by bacteria cultivated at 25°C were shown to be more soluble than those produced at 37°C and their dissociation by application of 2.4kbar at 20°C was shown to be further enhanced at −9°C. A red shift in intrinsic fluorescence spectra and an increase in binding of the hydrophobic fluorescent probe bis-ANS show subtle changes in conformation of ES in the presence of 1.5M GdnHCl at 2.4kbar, while at 0.4kbar the native conformational state is favored. The 25% refolding yield obtained via compression of IBs produced at 37°C by application of 2.4kbar, was increased to 78% when conditions based on the insights acquired were utilized: dissociation at 2.4kbar and −9°C of the IBs produced at 25°C, followed by refolding at 0.4kbar and 20°C. Besides providing insights into the conformational transitions of ES structure under HHP, this work proposes innovative conditions that are likely to have wide applicability to the HHP-induced refolding of proteins in general.

Structural alteration of Escherichia coli Hsp31 by thermal unfolding increases chaperone activity

Escherichia coli Hsp31, encoded by hchA, is a heat-inducible molecular chaperone. We found that Hsp31 undergoes a conformational change via temperature-induced unfolding, generating a high molecular weight (HMW) form with enhanced chaperone activity. Although it has previously been reported that some subunits of the Hsp31 crystal structure show structural heterogeneity with increased hydrophobic surfaces, Hsp31 basically forms a dimer. We found that a C-terminal deletion (CΔ19) of Hsp31 exhibited structurally and functionally similar characteristics to that of the HMW form. Both the CΔ19 and HMW forms achieved a structure with considerably more β-sheets and less α-helices than the native dimeric form, exposing a portion of its hydrophobic surfaces. The structural alterations were determined from its spectral changes in circular dichroism, intrinsic fluorescence of tryptophan residues, and fluorescence of bis-ANS binding to a hydrophobic surface. Interestingly, during thermal transition, the dimeric Hsp31 undergoes a conformational change to the HMW species via the CΔ19 structure, as monitored with near-UV CD spectrum, implying that the CΔ19 resembles an intermediate state between the dimer and the HMW form. From these results, we propose that Hsp31 transforms itself into a fully functional chaperone by altering its tertiary and quaternary structures.

Insights in small Heat Shock Protein/client interaction by combined protection analysis of two different client proteins

sHSPs interact with clients under denaturing conditions. CPH1Δ2, a truncated version of cyanobacterial phytochrome CPH1, was introduced as a new reporter (client). Comparative analyses of At17.8 and At17.6B as cytosolic class I sHSP representatives demonstrated the advantages of a chromophore-bearing photoreversible protein as new client for analyzing sHSP holdase function in addition to malate dehydrogenase (MDH). The tested sHSPs protected both clients in similar ways but with different efficiencies. Bis-ANS binding studies with sHSPs suggested that the bis-ANS binding is dependent on interactions between different sHSPs and MDH under denaturing temperatures. Structured summary of protein interactionsCPH1Δ2 and CPH1Δ2bind by molecular sieving (View interaction)CPH1Δ2 and HSP17.8bind by molecular sieving (View interaction)CPH1Δ2 and HSP17.6bind by molecular sieving (View interaction)MDH and HSP17.8bind by molecular sieving (View interaction)MDH and HSP17.6Bbind by molecular sieving (View interaction)

Engineering of an Environmentally Responsive Beta Roll Peptide for Use As a Calcium-Dependent Cross-Linking Domain for Peptide Hydrogel Formation

We have created a set of rationally designed peptides that form calcium-dependent hydrogels based on the beta roll peptide domain. In the absence of calcium, the beta roll domain is intrinsically disordered. Upon the addition of calcium, the peptide forms a beta helix secondary structure. We have designed two variations of our beta roll domain. First, we have mutated one face of the beta roll domain to contain leucine residues so that the calcium-dependent structural formation leads to dimerization through hydrophobic interactions. Second, an α-helical leucine zipper domain is appended to the engineered beta roll domain as an additional means of forming intermolecular cross-links. This full peptide construct forms a hydrogel only in calcium-rich environments. The resulting structural and mechanical properties of the supramolecular assemblies are compared with the wild-type domain using several biophysical techniques including circular dichroism, FRET, bis-ANS binding and microrheology. The calcium responsiveness and rheological properties of the leucine beta roll containing construct confirm the potential of this allosterically regulated scaffold to serve as a cross-linking domain for stimulus-responsive biomaterials development.

HspB2/Myotonic Dystrophy Protein Kinase Binding Protein (MKBP) as a Novel Molecular Chaperone: Structural and Functional Aspects

The small heat shock protein, human HspB2, also known as Myotonic Dystrophy Kinase Binding Protein (MKBP), specifically associates with and activates Myotonic Dystrophy Protein Kinase (DMPK), a serine/threonine protein kinase that plays an important role in maintaining muscle structure and function. The structure and function of HspB2 are not well understood. We have cloned and expressed the protein in E.coli and purified it to homogeneity. Far-UV circular dichroic spectrum of the recombinant HspB2 shows a β-sheet structure. Fluorescence spectroscopic studies show that the sole tryptophan residue at the 130th position is almost completely solvent-exposed. Bis-ANS binding shows that though HspB2 exhibits accessible hydrophobic surfaces, it is significantly less than that exhibited by another well characterized small HSP, αB-crystallin. Sedimentation velocity measurements show that the protein exhibits concentration-dependent oligomerization. Fluorescence resonance energy transfer study shows that HspB2 oligomers exchange subunits. Interestingly, HspB2 exhibits target protein-dependent chaperone-like activity: it exhibits significant chaperone-like activity towards dithiothreitol (DTT)-induced aggregation of insulin and heat-induced aggregation of alcohol dehydrogenase, but only partially prevents the heat-induced aggregation of citrate synthase, co-precipitating with the target protein. It also significantly prevents the ordered amyloid fibril formation of α-synuclein. Thus, our study, for the first time, provides biophysical characterization on the structural aspects of HspB2, and shows that it exhibits target protein-dependent chaperone-like activity.

Increased Hydrophobicity and Decreased Backbone Flexibility Explain the Lower Solubility of a Cataract-Linked Mutant of γD-Crystallin

A number of point mutations in γD-crystallin are associated with human cataract. The Pro23-to-Thr (P23T) mutation is perhaps the most common, is geographically widespread, and presents itself in a variety of phenotypes. It is therefore important to understand the molecular basis of lens opacity due to this mutation. In our earlier studies, we noted that P23T shows retrograde and sharply lowered solubility, most likely due to the emergence of hydrophobic patches involved in protein aggregation. Binding of 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonate (Bis-ANS) dye (a probe commonly used for detecting surface hydrophobicity) competed with aggregation, suggesting that the residues involved in Bis-ANS binding are also involved in protein aggregation. Here, using NMR spectroscopy in conjunction with Bis-ANS binding, we identify three residues (Y16, D21, and Y50) in P23T that are involved in binding the dye. Furthermore, using 15N NMR relaxation experiments, we show that, in the mutant protein, backbone fluctuations are restricted to the picosecond-to-nanosecond and microsecond timescales relative to the wild type. Our present studies specify the residues involved in these two pivotal characteristics of the mutant protein, namely increased surface hydrophobicity and restricted mobility of the protein backbone, which can explain the nucleation and further propagation of protein aggregates. Thus, we have now identified the residues in the P23T mutant that give rise to novel hydrophobic surfaces, as well as those regions of the protein backbone where fluctuations in different timescales are restricted, providing a comprehensive understanding of how lens opacity could result from this mutation.