Solvent-exposed Hydrophobic Surface Research Articles

Mechanistic insights into monomer level prevention of amyloid aggregation of lysozyme by glycyrrhizic acid

As the primary bioactive compound of glycyrrhiza rhizome, the triterpene glycoside conjugate Glycyrrhizic acid (GA) has demonstrated neuroprotective effects in vivo. This study evaluates the effectiveness of GA as an inhibitor of GuHCl-induced amyloid aggregation of hen egg white lysozyme (HEWL). Fibril formation as measured by Thioflavin-T fluorescence, 900 light scattering, and 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence illustrated ∼90 % prevention of fibrils at [GA]/[HEWL] ≥2:1. Images of Transmission electron microscopy evidence for the absence of any fibril or amorphous aggregation products. The spectral characteristics of soluble HEWL were in close resemblance to unfolded monomer. Computational and fluorescence investigations performed to analyse GA-HEWL interactions demonstrated slightly higher affinity of GA to unfolded HEWL and aggregation-prone regions. The likely mechanism of monomer level aggregation prevention by GA as dermined by computational, stability, and ANS experiments illustrated that GA modulated the compactness, solvent-accessible surface, and solvent-exposed hydrophobic surfaces of aggregation-prone state of HEWL. Our findings corroborate GA as an effective inhibitor of HEWL amyloid formation. To our knowledge, GA interaction-induced inhibition of aggregation-prone states has not been previously discussed. GA's modulation of aggregation-prone states of disease-related proteins will successfully develop GA as an amyloid inhibitor for clinical trials of amyloidosis and neurodegenerative illnesses.

Structure, chaperone activity, and aggregation of wild-type and R12C mutant αB-crystallins in the presence of thermal stress and calcium ion – Implications for role of calcium in cataract pathogenesis

The current study was performed with the aim to evaluate the chaperoning ability, structural features, and aggregation propensity of wild-type and R12C mutant αB-crystallins (αB-Cry) under thermal stress and in the presence of calcium ion. The results of different spectroscopic analyses suggest that wild-type and mutant αB-Cry have dissimilar secondary and tertiary structures. Moreover, αB-Cry indicates slightly improved chaperone activity upon the R12C mutation. Thermal stress and calcium, respectively, enhance and reduce the extent of solvent-exposed hydrophobic surfaces accompanying formation of ordered and non-ordered aggregate entities in both proteins. Compared to the wild-type protein, the R12C mutant counterpart shows significant resistance against thermal and calcium-induced aggregation. In addition, in the presence of calcium, significant structural variation was accompanied by reduction in the solvent-exposed hydrophobic patches and attenuation of chaperone activity in both proteins. Additionally, gel mobility shift assay indicates the intrinsic propensity of R12C mutant αB-Cry for disulfide bridge-mediated protein dimerization. Overall, the results of this study are of high significance for understanding the molecular details of different factors that are involved in the pathomechanism of cataract disorders.

Structure based aggregation studies reveal the presence of helix-rich intermediate during α-Synuclein aggregation.

Mechanistic understanding of nucleation dependent polymerization by α-synuclein (α-Syn) into toxic oligomers and amyloids is important for the drug development against Parkinson's disease. However the structural and morphological characterization during nucleation and subsequent fibrillation process of α-Syn is not clearly understood. Using a variety of complementary biophysical techniques monitoring entire pathway of nine different synucleins, we found that transition of unstructured conformation into β-sheet rich fibril formation involves helix-rich intermediates. These intermediates are common for all aggregating synucleins, contain high solvent-exposed hydrophobic surfaces, are cytotoxic to SHSY-5Y cells and accelerate α-Syn aggregation efficiently. A multidimensional NMR study characterizing the intermediate accompanied with site-specific fluorescence study suggests that the N-terminal and central portions mainly participate in the helix-rich intermediate formation while the C-terminus remained in an extended conformation. However, significant conformational transitions occur at the middle and at the C-terminus during helix to β-sheet transition as evident from Trp fluorescence study. Since partial helix-rich intermediates were also observed for other amyloidogenic proteins such as Aβ and IAPP, we hypothesize that this class of intermediates may be one of the important intermediates for amyloid formation pathway by many natively unstructured protein/peptides and represent a potential target for drug development against amyloid diseases.

Solution NMR Structure of the Ca2+-bound N-terminal Domain of CaBP7

Calcium-binding protein 7 (CaBP7) is a member of the calmodulin (CaM) superfamily that harbors two high affinity EF-hand motifs and a C-terminal transmembrane domain. CaBP7 has been previously shown to interact with and modulate phosphatidylinositol 4-kinase III-β (PI4KIIIβ) activity in in vitro assays and affects vesicle transport in neurons when overexpressed. Here we show that the N-terminal domain (NTD) of CaBP7 is sufficient to mediate the interaction of CaBP7 with PI4KIIIβ. CaBP7 NTD encompasses the two high affinity Ca(2+) binding sites, and structural characterization through multiangle light scattering, circular dichroism, and NMR reveals unique properties for this domain. CaBP7 NTD binds specifically to Ca(2+) but not Mg(2+) and undergoes significant conformational changes in both secondary and tertiary structure upon Ca(2+) binding. The Ca(2+)-bound form of CaBP7 NTD is monomeric and exhibits an open conformation similar to that of CaM. Ca(2+)-bound CaBP7 NTD has a solvent-exposed hydrophobic surface that is more expansive than observed in CaM or CaBP1. Within this hydrophobic pocket, there is a significant reduction in the number of methionine residues that are conserved in CaM and CaBP1 and shown to be important for target recognition. In CaBP7 NTD, these residues are replaced with isoleucine and leucine residues with branched side chains that are intrinsically more rigid than the flexible methionine side chain. We propose that these differences in surface hydrophobicity, charge, and methionine content may be important in determining highly specific interactions of CaBP7 with target proteins, such as PI4KIIIβ.

A systematic study of fundamentals in α-helical coiled coil mimicry by alternating sequences of β- and γ-amino acids

Aimed at understanding the crucially important structural features for the integrity of α-helical mimicry by βγ-sequences, an α-amino acid sequence in a native peptide was substituted by differently arranged βγ-sequences. The self- and hetero-assembly of a series of αβγ-chimeric sequences based on a 33-residue GCN4-derived peptide was investigated by means of molecular dynamics, circular dichroism, and a disulfide exchange assay. Despite the native-like behavior of βγ alternating sequences such as retention of α-helix dipole and the formation of 13-membered α-helix turns, the αβγ-chimeras with different βγ substitution patterns do not equally mimic the structural behavior of the native parent peptide in solution. The preservation of the key residue contacts such as van der Waals interactions and intrahelical H-bonding, which can be met only by particular substitution patterns, thermodynamically favor the adoption of coiled coil folding motif. In this study, we show how successfully the destabilizing structural consequences of α→βγ modification can be harnessed by reducing the solvent-exposed hydrophobic surface area and placing of suitably long and bulky helix-forming side chains at the hydrophobic core. The pairing of αβγ-chimeric sequences with the native wild-type are thermodynamically allowed in the case of ideal arrangement of β- and γ-residues. This indicates a similarity in local side chain packing of β- and γ-amino acids at the helical interface of αβγ-chimeras and the native α-peptide. Consequently, the backbone extended residues are able to participate in classical "knob-into-hole" packing with native α-peptide.

Gradual Adaptive Changes of a Protein Facing High Salt Concentrations

Several experimental techniques were applied to unravel fine molecular details of protein adaptation to high salinity. We compared four homologous enzymes, which suggested a new halo-adaptive state in the process of molecular adaptation to high-salt conditions. Together with comparative functional studies, the structure of malate dehydrogenase from the eubacterium Salinibacter ruber shows that the enzyme shares characteristics of a halo-adapted archaea-bacterial enzyme and of non-halo-adapted enzymes from other eubacterial species. The S. ruber enzyme is active at the high physiological concentrations of KCl but, unlike typical halo-adapted enzymes, remains folded and active at low salt concentrations. Structural aspects of the protein, including acidic residues at the surface, solvent-exposed hydrophobic surface, and buried hydrophobic surface, place it between the typical halo-adapted and non-halo-adapted proteins. The enzyme lacks inter-subunit ion-binding sites often seen in halo-adapted enzymes. These observations permit us to suggest an evolutionary pathway that is highlighted by subtle trade-offs to achieve an optimal compromise among solubility, stability, and catalytic activity.

Open and closed conformations of two SpoIIAA-like proteins (YP_749275.1 and YP_001095227.1) provide insights into membrane association and ligand binding.

The crystal structures of the proteins encoded by the YP_749275.1 and YP_001095227.1 genes from Shewanella frigidimarina and S. loihica, respectively, have been determined at 1.8 and 2.25 Å resolution, respectively. These proteins are members of a novel family of bacterial proteins that adopt the α/β SpoIIAA-like fold found in STAS and CRAL-TRIO domains. Despite sharing 54% sequence identity, these two proteins adopt distinct conformations arising from different dispositions of their α2 and α3 helices. In the `open' conformation (YP_001095227.1), these helices are 15 Å apart, leading to the creation of a deep nonpolar cavity. In the `closed' structure (YP_749275.1), the helices partially unfold and rearrange, occluding the cavity and decreasing the solvent-exposed hydrophobic surface. These two complementary structures are reminiscent of the conformational switch in CRAL-TRIO carriers of hydrophobic compounds. It is suggested that both proteins may associate with the lipid bilayer in their `open' monomeric state by inserting their amphiphilic helices, α2 and α3, into the lipid bilayer. These bacterial proteins may function as carriers of nonpolar substances or as interfacially activated enzymes.

Self-assembly of Bacteriophage-associated Hyaluronate Lyase (HYLP2) into an Enzymatically Active Fibrillar Film

The in vitro assembly of a soluble protein into its mature fibrillar form is usually accompanied by loss of its functional activity. Our study is the first demonstration of a natural enzyme (HylP2) retaining its enzymatic activity on conversion from pre-fibril to mature fibril and supports the contention that minor conformational changes in the native folded form of a protein can lead to the formation of a functional fibril. Hyaluronate lyase (HylP2) is a natural enzyme of bacteriophage 10403 of Streptococcus pyogenes. At pH 5.0, the enzyme undergoes partial unfolding localized in its N-terminal domain while the C-terminal domain maintains its folded trimeric conformation. This structural variant of HylP2 retains about 70% enzymatic activity with hyaluronan. It further self-assembles into a fibrillar film in vitro through solvent-exposed nonpolar surfaces and intermolecular beta-sheet formation by the beta-strands in the protein. Interestingly, the mature fibrillar film of HylP2 also retains about 60 and 20% enzymatic activity for hyaluronic acid and chondroitin sulfate, respectively. The possession of broad substrate specificity by the fibrillar form of HylP2 indicates that fluctuations in pH, which do not lead to loss of functionality of HylP2, might assist in bacterial pathogenesis. The formation of fibrillar film-like structure has been observed for the first time among the hyaluronidase enzymes. After acquiring this film-like structure in bacteriophage, HylP2 still retains its enzymatic activity, which establishes that these fibrils are a genuinely acquired protein fold/structure.

Interactions Defined between S100A13 and Annexin Peptides: Insight into Non-Classical Secrection

S100A13 is a calcium binding chaperone protein, known to be involved in the non-classical export of signal peptide less proteins such as fibroblast growth factor (FGF-1) and interleukin-1α across the cell membrane. It has also been shown that the interaction of S100A13 with Annexin II, which exhibits an inducible flip-flop mechanism across the cell bilayer, helps the multiprotein release complex to traverse the membrane bilayer. The interaction of S100A13 and annexin II has been characterized using various biophysical techniques including multidimensional NMR spectroscopy. Results of the Isothermal titration calorimetry (ITC) experiments show that holo-S100A13 exhibits preferential binding to annexin II with high affinity in the micro molar rang compared to apo-S100A13. Equilibrium guanidine hydrochloride denaturation monitored by steady-state fluorescence and limited trypsin digestion analysis reveals holo-S100A13 is significantly stabilized upon binding to annexin II peptide compared to apo-S100A13. ANS (8-anilino-1-napthalene sulfonate) binding experiments indicates that the presence of annexin II peptide does not increase the solvent availability of hydrophobic residues in holo-S100A13. Availability of the solvent-exposed hydrophobic surface(s) in apoS10013 does not facilitate its interaction with the annexin II peptide, which is unique characteristic of S100A13. 1H-15N- HSQC NMR experiments reveal that the binding site of annexin II peptide on holo-S100A13 is modestly different from other S100/Annexin interactions. S100 proteins typically interact with more than one Annexin protein. In order to define the specificity of S100A13 for Annexin 2, the interaction of S100A13 with other Annexin peptides was characterized and data characterizing these interactions will be presented. Finally, defining the interaction between S100A13 and Annexin 2 peptide will give much needed insight into the non-classical release of the signal peptide-less protein Fibroblast Growth Factor-1

Formation of an Unfolding Intermediate State of Soluble Chloride Intracellular Channel Protein CLIC1 at Acidic pH

CLIC proteins function as anion channels when their structures convert from a soluble form to an integral membrane form. While very little is known about the mechanism of the conversion process, channel formation and activity are highly pH-dependent. In this study, the structural properties and conformational stability of CLIC1 were determined as a function of pH in the absence of membranes to improve our understanding of how its conformation changes when the protein encounters the acidic environment at the surface of a membrane. Although the global conformation and size of CLIC1 are not significantly altered by pH in the range of 5.5-8.2, equilibrium unfolding studies reveal that the protein molecule becomes destabilized at low pH, resulting in the formation of a highly populated intermediate with a solvent-exposed hydrophobic surface. Unlike the intermediates formed by many soluble pore-forming proteins for their insertion into membranes, the CLIC1 intermediate is not a molten globule. Acid-induced destabilization and partial unfolding of CLIC1 involve helix alpha1 which is the major structural element of the transmembrane region. We propose that the acidic environment encountered by CLICs at the surface of membranes primes the transmembrane region in the N-domain, thereby lowering the energy barrier for the conversion of soluble CLICs to their membrane-inserted forms.

A Structural Model of the GDP Dissociation Inhibitor Rab Membrane Extraction Mechanism

Rab GDP dissociation inhibitors (GDI)-facilitated extraction of prenylated Rab proteins from membranes plays an important role in vesicular membrane trafficking. The investigated thermodynamic properties of yeast Rab.GDI and Rab.MRS6 complexes demonstrated differences in the Rab binding properties of the closely related Rab GDI and MRS6 proteins, consistent with their functional diversity. The importance of the Rab C terminus and its prenylation for GDI/MRS6 binding was demonstrated using both biochemical and structural data. The presented structures of the apo-form yeast Rab GDI and its two complexes with unprenylated Rab proteins, together with the earlier published structures of the prenylated Ypt1.GDI, provide evidence of allosteric regulation of the GDI lipid binding site opening, which plays a key role in the proposed mechanism of GDI-mediated Rab extraction. We suggest a model for the interaction of GDI with prenylated Rab proteins that incorporates a stepwise increase in affinity as the three different partial interactions are successively formed.

S100A13–lipid interactions—role in the non-classical release of the acidic fibroblast growth factor

S100A13 is a 98-amino acid, calcium binding protein. It is known to participate in the non-classical secretion of signal peptide-less proteins, such as the acidic fibroblast growth factor. In this study, we investigate the lipid binding properties of S10013 using a number of biophysical techniques, including multidimensional NMR spectroscopy. Isothermal titration calorimetry and steady state fluorescence experiments show that apoS100A13 exhibits preferential binding to small unilamelar vesicles of l-phosphatidyl serine (pS). In comparison, Ca 2+-bound S100A13 is observed to bind weakly to unilamelar vesicles (SUVs) of pS. Equilibrium thermal unfolding and limited trypsin digestion analysis reveal that apoS100A13 is significantly destabilized upon binding to SUVs of pS. Results of the far UV circular dichroism and ANS (8-anilino-1-napthalene sufonate) binding experiments indicate a subtle conformational change resulting in the increase in the solvent-accessible hydrophobic surface in the protein. Availability of the solvent-exposed hydrophobic surface(s) in apoS10013 facilitates its interaction with the lipid vesicles. Our data suggest that Ca 2+ binding dictates the membrane binding affinity of S100A13. Based on the results of this study, a model describing the sequence of molecular events that possibly can occur during the non-classical secretion of FGF-1 is presented.

Structural Reorganisation and Potential Toxicity of Oligomeric Species Formed during the Assembly of Amyloid Fibrils

Increasing evidence indicates that oligomeric protein assemblies may represent the molecular species responsible for cytotoxicity in a range of neurological disorders including Alzheimer and Parkinson diseases. We use all-atom computer simulations to reveal that the process of oligomerization can be divided into two steps. The first is characterised by a hydrophobic coalescence resulting in the formation of molten oligomers in which hydrophobic residues are sequestered away from the solvent. In the second step, the oligomers undergo a process of reorganisation driven by interchain hydrogen bonding interactions that induce the formation of β sheet rich assemblies in which hydrophobic groups can become exposed. Our results show that the process of aggregation into either ordered or amorphous species is largely determined by a competition between the hydrophobicity of the amino acid sequence and the tendency of polypeptide chains to form arrays of hydrogen bonds. We discuss how the increase in solvent-exposed hydrophobic surface resulting from such a competition offers an explanation for recent observations concerning the cytotoxicity of oligomeric species formed prior to mature amyloid fibrils.

Analysis of acidic surface of Haloferax mediterranei glucose dehydrogenase by site-directed mutagenesis

Generally, halophilic enzymes present a characteristic amino acid composition, showing an increase in the content of acidic residues and a decrease in the content of basic residues, particularly lysines. The latter decrease appears to be responsible for a reduction in the proportion of solvent-exposed hydrophobic surface. This role was investigated by site-directed mutagenesis of glucose dehydrogenase from Haloferax mediterranei, in which surface aspartic residues were changed to lysine residues. From the biochemical analysis of the mutant proteins, it is concluded that the replacement of the aspartic residues by lysines results in slightly less halotolerant proteins, although they retain the same enzymatic activities and kinetic parameters compared to the wild type enzyme.

Distinct Early Folding and Aggregation Properties of Alzheimer Amyloid-β Peptides Aβ40 and Aβ42: STABLE TRIMER OR TETRAMER FORMATION BY Aβ42

The amyloid beta peptide (Abeta), composed of 40 or 42 amino acids, is a critical component in the etiology of the neurodegenerative Alzheimer disease. Abeta is prone to aggregate and forms amyloid fibrils progressively both in vitro and in vivo. To understand the process of amyloidogenesis, it is pivotal to examine the initial stages of the folding process. We examined the equilibrium folding properties, assembly states, and stabilities of the early folding stages of Abeta40 and Abeta42 prior to fibril formation. We found that Abeta40 and Abeta42 have different conformations and assembly states upon refolding from their unfolded ensembles. Abeta40 is predominantly an unstable and collapsed monomeric species, whereas Abeta42 populates a stable structured trimeric or tetrameric species at concentrations above approximately 12.5 microm. Thermodynamic analysis showed that the free energies of Abeta40 monomer and Abeta42 trimer/tetramer are approximately 1.1 and approximately 15/ approximately 22 kcal/mol, respectively. The early aggregation stages of Abeta40 and Abeta42 contain different solvent-exposed hydrophobic surfaces that are located at the sequences flanking its protease-resistant segment. The amyloidogenic folded structure of Abeta is important for the formation of spherical beta oligomeric species. However, beta oligomers are not an obligatory intermediate in the process of fibril formation because oligomerization is inhibited at concentrations of urea that have no effect on fibril formation. The distinct initial folding properties of Abeta40 and Abeta42 may play an important role in the higher aggregation potential and pathological significance of Abeta42.

PDZ7 of Glutamate Receptor Interacting Protein Binds to Its Target via a Novel Hydrophobic Surface Area

Glutamate receptor interacting protein 1 (GRIP1) is a scaffold protein composed of seven PDZ (Postsynaptic synaptic density-95/Discs large/Zona occludens-1) domains. The protein plays important roles in the synaptic targeting of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The interaction between GRIP1 PDZ7 and a Ras guanine nucleotide exchange factor, GRASP-1, regulates synaptic distribution of AMPA receptors. Here, we describe the three-dimensional structure of GRIP1 PDZ7 determined by NMR spectroscopy. GRIP1 PDZ7 contains a closed carboxyl group-binding pocket and a narrow alphaB/betaB-groove that is not likely to bind to classical PDZ ligands. Unexpectedly, GRIP1 PDZ7 contains a large solvent-exposed hydrophobic surface at a site distinct from the conventional ligand-binding alphaB/betaB-groove. NMR titration experiments show that GRIP1 PDZ7 binds to GRASP-1 via this hydrophobic surface. Our data uncover a novel PDZ domain-mediated protein interaction mode that may be responsible for multimerization of other PDZ domain-containing scaffold proteins.

Specificity in intracellular protein aggregation and inclusion body formation.

Protein aggregation is widely considered to be a nonspecific coalescence of misfolded proteins, driven by interactions between solvent-exposed hydrophobic surfaces that are normally buried within a protein's interior. Accordingly, abnormal interactions between misfolded proteins with normal cellular constituents has been proposed to underlie the toxicity associated with protein aggregates in many neurodegenerative disorders. Here we have used fluorescence resonance energy transfer and deconvolution microscopy to investigate the degree to which unrelated misfolded proteins expressed in the same cells coaggregate with one another. Our data reveal that in cells, protein aggregation exhibits exquisite specificity even among extremely hydrophobic substrates expressed at very high levels.

The hairpin structure of the (6)F1(1)F2(2)F2 fragment from human fibronectin enhances gelatin binding.

The solution structure of the (6)F1(1)F2(2)F2 fragment from the gelatin-binding region of fibronectin has been determined (Protein Data Bank entry codes 1e88 and 1e8b). The structure reveals an extensive hydrophobic interface between the non-contiguous (6)F1 and (2)F2 modules. The buried surface area between (6)F1 and (2)F2 ( approximately 870 A(2)) is the largest intermodule interface seen in fibronectin to date. The dissection of (6)F1(1)F2(2)F2 into the (6)F1(1)F2 pair and (2)F2 results in near-complete loss of gelatin-binding activity. The hairpin topology of (6)F1(1)F2(2)F2 may facilitate intramolecular contact between the matrix assembly regions flanking the gelatin-binding domain. This is the first high-resolution study to reveal a compact, globular arrangement of modules in fibronectin. This arrangement is not consistent with the view that fibronectin is simply a linear 'string of beads'.

Molecular Chaperone-like Properties of an Unfolded Protein, αs-Casein

All molecular chaperones known to date are well organized, folded protein molecules whose three-dimensional structure are believed to play a key role in the mechanism of substrate recognition and subsequent assistance to folding. A common feature of all protein and nonprotein molecular chaperones is the propensity to form aggregates very similar to the micellar aggregates. In this paper we show that alpha(s)-casein, abundant in mammalian milk, which has no well defined secondary and tertiary structure but exits in nature as a micellar aggregate, can prevent a variety of unrelated proteins/enzymes against thermal-, chemical-, or light-induced aggregation. It also prevents aggregation of its natural substrates, the whey proteins. alpha(s)-Casein interacts with partially unfolded proteins through its solvent-exposed hydrophobic surfaces. The absence of disulfide bridge or free thiol groups in its sequence plays important role in preventing thermal aggregation of whey proteins caused by thiol-disulfide interchange reactions. Our results indicate that alpha(s)-casein not only prevents the formation of huge insoluble aggregates but it can also inhibit accumulation of soluble aggregates of appreciable size. Unlike other molecular chaperones, this protein can solubilize hydrophobically aggregated proteins. This protein seems to have some characteristics of cold shock protein, and its chaperone-like activity increases with decrease of temperature.

Solution structure of a protein inhibitor of neuronal nitric oxide synthase

The structure of the neuronal nitric oxide synthase inhibitory protein, PIN (protein inhibitor of nNOS), has been determined by NMR spectroscopy. Two N-terminal antiparallel alpha-helices pack against a four-stranded antiparallel beta-sheet in the C-terminal region of the protein, forming a two-layer alpha/beta plait. The three dimensional structure of PIN resembles the fold of the B-chain of aspartylglucosaminidase. A non-prolyl cis peptide bond was found between Pro 52 and Thr 53 of the protein. PIN has a large solvent-exposed hydrophobic surface that contains a cavity and is rimmed with positive charges. This surface may serve as the primary target-binding region for this multi-functional regulatory protein.