Probing folding and fluorescence quenching in human gammaD crystallin Greek key domains using triple tryptophan mutant proteins

Human gammaD crystallin (HgammaD-Crys), a major component of the human eye lens, is a 173-residue, primarily beta-sheet protein, associated with juvenile and mature-onset cataracts. HgammaD-Crys has four tryptophans, with two in each of the homologous Greek key domains, which are conserved throughout the gamma-crystallin family. HgammaD-Crys exhibits native-state fluorescence quenching, despite the absence of ligands or cofactors. The tryptophan absorption and fluorescence quenching may influence the lens response to ultraviolet light or the protection of the retina from ambient ultraviolet damage. To provide fluorescence reporters for each quadrant of the protein, triple mutants, each containing three tryptophan-to-phenylalanine substitutions and one native tryptophan, have been constructed and expressed. Trp 42-only and Trp 130-only exhibited fluorescence quenching between the native and denatured states typical of globular proteins, whereas Trp 68-only and Trp 156-only retained the anomalous quenching pattern of wild-type HgammaD-Crys. The three-dimensional structure of HgammaD-Crys shows Tyr/Tyr/His aromatic cages surrounding Trp 68 and Trp 156 that may be the source of the native-state quenching. During equilibrium refolding/unfolding at 37 degrees C, the tryptophan fluorescence signals indicated that domain I (W42-only and W68-only) unfolded at lower concentrations of GdnHCl than domain II (W130-only and W156-only). Kinetic analysis of both the unfolding and refolding of the triple-mutant tryptophan proteins identified an intermediate along the HgammaD-Crys folding pathway with domain I unfolded and domain II intact. This species is a candidate for the partially folded intermediate in the in vitro aggregation pathway of HgammaD-Crys.     

Interdomain side-chain interactions in human gammaD crystallin influencing folding and stability

Human gammaD crystallin (HgammaD-Crys) is a two domain, beta-sheet eye lens protein that must remain soluble throughout life for lens transparency. Single amino acid substitutions of HgammaD-Crys are associated with juvenile-onset cataracts. Features of the interface between the two domains conserved among gamma-crystallins are a central six-residue hydrophobic cluster, and two pairs of interacting residues flanking the cluster. In HgammaD-Crys these pairs are Gln54/Gln143 and Arg79/Met147. We previously reported contributions of the hydrophobic cluster residues to protein stability. In this study alanine substitutions of the flanking residue pairs were constructed and analyzed. Equilibrium unfolding/refolding experiments at 37 degrees C revealed a plateau in the unfolding/refolding transitions, suggesting population of a partially folded intermediate with a folded C-terminal domain (C-td) and unfolded N-terminal domain (N-td). The N-td was destabilized by substituting residues from both domains. In contrast, the C-td was not significantly affected by substitutions of either domain. Refolding rates of the N-td were significantly decreased for mutants of either domain. In contrast, refolding rates of the C-td were similar to wild type for mutants of either domain. Therefore, domain interface residues of the folded C-td probably nucleate refolding of the N-td. We suggest that these residues stabilize the native state by shielding the central hydrophobic cluster from solvent. Glutamine and methionine side chains are among the residues covalently damaged in aged and cataractous lenses. Such damage may generate partially unfolded, aggregation- prone conformations of HgammaD-Crys that could be significant in cataract.     

Reversible unfolding and refolding behavior of a monomeric aldolase from Staphylococcus aureus.

Thermal and GdmCl-induced unfolding transitions of aldolase from Staphylococcus aureus are reversible under a variety of solvent conditions. Analysis of the transitions reveals that no partially folded intermediates can be detected under equilibrium conditions. The stability of the enzyme is very low with a delta G0 value of -9 +/- 2 kJ/mol at 20 degrees C. The kinetics of unfolding and refolding of aldolase are complex and comprise at least one fast and two slow reactions. This complexity arises from prolyl isomerization reactions in the unfolded chain, which are kinetically coupled to the actual folding reaction. Comparison with model calculations shows that at least two prolyl peptide bonds give rise to the observed slow folding reactions of aldolase and that all of the involved bonds are presumably in the trans conformation in the native state. The rate constant of the actual folding reaction is fast with a relaxation time of about 15 s at the midpoint of the folding transition at 15 degrees C. The data presented on the folding and stability of aldolase are comparable to the properties of much smaller proteins. This might be connected with the simple and highly repetitive tertiary structure pattern of the enzyme, which belongs to the group of alpha/beta barrel proteins.     

Cofactor effects on the protein folding reaction: acceleration of alpha-lactalbumin refolding by metal ions

About 30% of proteins require cofactors for their proper folding. The effects of cofactors on the folding reaction have been investigated with alpha-lactalbumin as a model protein and metal ions as cofactors. Metal ions accelerate the refolding of alpha-lactalbumin by lessening the energy barrier between the molten globule state and the transition state, mainly by decreasing the difference of entropy between the two states. These effects are linked to metal ion binding to the protein in the native state. Hence, relationships between the metal affinities for the intermediate states and those for the native state are observed. Some residual specificity for the calcium ion is still observed in the molten globule state, this specificity getting closer in the transition state to that of the native state. The comparison between kinetic and steady-state data in association with the Phi value method indicates the binding of the metal ions on the unfolded state of alpha-lactalbumin. Altogether, these results provide insight into cofactor effects on protein folding. They also suggest new possibilities to investigate the presence of residual native structures in the unfolded state of protein and the effects of such structures on the protein folding reaction and on protein stability.     

Thermal folding and mechanical unfolding pathways of protein secondary structures

Mechanical stretching of secondary structures is studied through molecular dynamics simulations of a Go-like model. Force versus displacement curves are studied as a function of the stiffness and velocity of the pulling device. The succession of stretching events, as measured by the order in which contacts are ruptured, is compared to the sequencing of events during thermal folding and unfolding. Opposite cross-correlations are found for an alpha-helix and a beta-hairpin structure. In a tandem of two alpha-helices, the two constituent helices unravel nearly simultaneously. A simple condition for simultaneous versus sequential unraveling of repeat units is presented.     

Insights into the domains required for dimerization and assembly of human alphaB crystallin

Protein pin array technology was used to identify subunit-subunit interaction sites in the small heat shock protein (sHSP) alphaB crystallin. Subunit-subunit interaction sites were defined as consensus sequences that interacted with both human alphaA crystallin and alphaB crystallin. The human alphaB crystallin protein pin array consisted of contiguous and overlapping peptides, eight amino acids in length, immobilized on pins that were in a 96-well ELISA plate format. The interaction of alphaB crystallin peptides with physiological partner proteins, alphaA crystallin and alphaB crystallin, was detected using antibodies and recorded using spectrophotometric absorbance. Five peptide sequences including 37LFPTSTSLSPFYLRPPSF54 in the N terminus, 75FSVNLDVK82)(beta3), 131LTITSSLS138 (beta8) and 141GVLTVNGP148 (beta9) that form beta strands in the conserved alpha crystallin core domain, and 155PERTIPITREEK166 in the C-terminal extension were identified as subunit-subunit interaction sites in human alphaB crystallin using the novel protein pin array assay. The subunit-subunit interaction sites were mapped to a three-dimensional (3D) homology model of wild-type human alphaB crystallin that was based on the crystal structure of wheat sHSP16.9 and Methanococcus jannaschi sHSP16.5 (Mj sHSP16.5). The subunit-subunit interaction sites identified and mapped onto the homology model were solvent-exposed and had variable secondary structures ranging from beta strands to random coils and short alpha helices. The subunit-subunit interaction sites formed a pattern of hydrophobic patches on the 3D surface of human alphaB crystallin.     

Urea modulation of beta-amyloid fibril growth: experimental studies and kinetic models

Aggregation of beta-amyloid (Abeta) into fibrillar deposits is widely believed to initiate a cascade of adverse biological responses associated with Alzheimer's disease. Although it was once assumed that the mature fibril was the toxic form of Abeta, recent evidence supports the hypothesis that Abeta oligomers, intermediates in the fibrillogenic pathway, are the dominant toxic species. In this work we used urea to reduce the driving force for Abeta aggregation, in an effort to isolate stable intermediate species. The effect of urea on secondary structure, size distribution, aggregation kinetics, and aggregate morphology was examined. With increasing urea concentration, beta-sheet content and the fraction of aggregated peptide decreased, the average size of aggregates was reduced, and the morphology of aggregates changed from linear to a globular/linear mixture and then to globular. The data were analyzed using a previously published model of Abeta aggregation kinetics. The model and data were consistent with the hypothesis that the globular aggregates were intermediates in the amyloidogenesis pathway rather than alternatively aggregated species. Increasing the urea concentration from 0.4 M to 2 M decreased the rate of filament initiation the most; between 2 M and 4 M urea the largest change was in partitioning between the nonamyloid and amyloid pathways, and between 4 M and 6 M urea, the most significant change was a reduction in the rate of filament elongation.     

BetaCore,a designed water soluble four-stranded antiparallel beta-sheet protein

BetaCore is a designed approximately 50-residue protein in which two BPTI-derived core modules, CM I and CM II, are connected by a 22-atom cross-link. At low temperature and pH 3, homo- and heteronuclear NMR data report a dominant folded ('f') conformation with well-dispersed chemical shifts, i, i+1 periodicity, numerous long-range NOEs, and slowed amide hydrogen isotope exchange patterns that is a four-stranded antiparallel beta-sheet with nonsymmetrical and specific association of CM I and CM II. BetaCore 'f' conformations undergo reversible, global, moderately cooperative, non-two-state thermal transitions to an equilibrium ensemble of unfolded 'u' conformations. There is a significant energy barrier between 'f' and 'u' conformations. This is the first designed four-stranded antiparallel beta-sheet that folds in water.     

Fluorescence resonance energy transfer study of subunit exchange in human lens crystallins and congenital cataract crystallin mutants

Lens α-crystallin is an oligomeric protein with a molecular mass of 500–1000 kDa and a polydispersed assembly. It consists of two types of subunits, αA and αB, each with a molecular mass of 20 kDa. The subunits also form homo-oligomers in some other tissues and in vitro. Their quaternary structures, which are dynamic and characterized by subunit exchange, have been studied by many techniques, including fluorescence resonance energy transfer (FRET) and mass spectrometry analysis. The proposed mechanism of subunit exchange has been either by dissociation/association of monomeric subunits or by rapid equilibrium between oligomers and suboligomers. To explore the nature of subunit exchange further, we performed additional FRET measurements and analyses using a fluorescent dye-labeled W9F αA-crystallin as the acceptor probe and Trp in other crystallins (wild-type and R116C αA, wild-type and R120G αB, wild-type and Q155* βB2) as the donor probe and calculated the transfer efficiency, Förster distance, and average distance between two probes. The results indicate only slight decreased efficiency and increased distance between two probes for the R116C αA and R120G αB mutations despite conformational changes.     

Deamidation destabilizes and triggers aggregation of a lens protein, betaA3-crystallin

Protein aggregation is a hallmark of several neurodegenerative diseases and also of cataracts. The major proteins in the lens of the eye are crystallins, which accumulate throughout life and are extensively modified. Deamidation is the major modification in the lens during aging and cataracts. Among the crystallins, the betaA3-subunit has been found to have multiple sites of deamidation associated with the insoluble proteins in vivo. Several sites were predicted to be exposed on the surface of betaA3 and were investigated in this study. Deamidation was mimicked by site-directed mutagenesis at Q42 and N54 on the N-terminal domain, N133 and N155 on the C-terminal domain, and N120 in the peptide connecting the domains. Deamidation altered the tertiary structure without disrupting the secondary structure or the dimer formation of betaA3. Deamidations in the C-terminal domain and in the connecting peptide decreased stability to a greater extent than deamidations in the N-terminal domain. Deamidation at N54 and N155 also disrupted the association with the betaB1-subunit. Sedimentation velocity experiments integrated with high-resolution analysis detected soluble aggregates at 15%-20% in all deamidated proteins, but not in wild-type betaA3. These aggregates had elevated frictional ratios, suggesting that they were elongated. The detection of aggregates in vitro strongly suggests that deamidation may contribute to protein aggregation in the lens. A potential mechanism may include decreased stability and/or altered interactions with other beta-subunits. Understanding the role of deamidation in the long-lived crystallins has important implications in other aggregation diseases.     

Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM,molecular dynamics simulations,NMR and protein engineering

The mechanical unfolding of an immunoglobulin domain from the human muscle protein titin (TI I27) has been shown to proceed via a metastable intermediate in which the A-strand is detached. The structure and properties of this intermediate are characterised in this study. A conservative destabilising mutation in the A-strand has no effect on the unfolding force, nor the dependence of the unfolding force on the pulling speed, indicating that the unfolding forces measured in an AFM experiment are those required for the unfolding of the intermediate and not the native state. A mutant of TI I27 with the A-strand deleted (TI I27-A) is studied by NMR and standard biophysical techniques, combined with protein engineering. Molecular dynamics simulations show TI I27-A to be a good model for the intermediate. It has a structure very similar to the native state, and is surprisingly stable. Comparison with a Phi-value analysis of the unfolding pathway clearly shows that the protein unfolds by a different pathway under an applied force than on addition of denaturant.     

The roles of turn formation and cross-strand interactions in fibrillization of peptides derived from the OspA single-layer beta-sheet

We previously demonstrated that a beta-hairpin peptide, termed BH(9-10), derived from a single-layer beta-sheet of Borrelia OspA protein, formed a native-like beta-turn in trifluoroethanol (TFE) solution, and it assembled into amyloid-like fibrils at higher TFE concentrations. This peptide is highly charged, and fibrillization of such a hydrophilic peptide is quite unusual. In this study, we designed a circularly permutated peptide of BH(9-10), termed BH(10-9). When folded into their respective beta-hairpin structures found in OspA, these peptides would have identical cross-strand interactions but different turns connecting the strands. NMR study revealed that BH(10-9) had little propensity to form a turn structure both in aqueous and TFE solutions. At higher TFE concentration, BH(10-9) precipitated with a concomitant alpha-to-beta conformational conversion, in a similar manner to the BH(9-10) fibrillization. However, the BH(10-9) precipitates were nonfibrillar aggregation. The precipitation kinetics of BH(10-9) was exponential, consistent with a first-order molecular assembly reaction, while the fibrillization of BH(9-10) showed sigmoidal kinetics, indicative of a two-step reaction consisting of nucleation and molecular assembly. The correlation between native-like turn formation and fibrillization of our peptide system strongly suggests that BH(9-10) adopts a native-like beta-hairpin conformation in the fibrils. Remarkably, seeding with the preformed BH(10-9) precipitates changed the two-step BH(9-10) fibrillization to a one-step molecular assembly reaction, and disrupted the BH(9-10) fibril structure, indicating interactions between the BH(10-9) aggregates and the BH(9-10) peptide. Our results suggest that, in these peptides, cross-strand interactions are the driving force for molecular assembly, and turn formation limits modes of peptide assembly.     

Factors involved in the stability of isolated beta-sheets: Turn sequence, beta-sheet twisting, and hydrophobic surface burial

We have recently reported on the design of a 20-residue peptide able to form a significant population of a three-stranded up-and-down antiparallel beta-sheet in aqueous solution. To improve our beta-sheet model in terms of the folded population, we have modified the sequences of the two 2-residue turns by introducing the segment DPro-Gly, a sequence shown to lead to more rigid type II' beta-turns. The analysis of several NMR parameters, NOE data, as well as Deltadelta(CalphaH), DeltadeltaC(beta), and Deltadelta(Cbeta) values, demonstrates that the new peptide forms a beta-sheet structure in aqueous solution more stable than the original one, whereas the substitution of the DPro residues by LPro leads to a random coil peptide. This agrees with previous results on beta-hairpin-forming peptides showing the essential role of the turn sequence for beta-hairpin folding. The well-defined beta-sheet motif calculated for the new designed peptide (pair-wise RMSD for backbone atoms is 0.5 +/- 0.1 A) displays a high degree of twist. This twist likely contributes to stability, as a more hydrophobic surface is buried in the twisted beta-sheet than in a flatter one. The twist observed in the up-and-down antiparallel beta-sheet motifs of most proteins is less pronounced than in our designed peptide, except for the WW domains. The additional hydrophobic surface burial provided by beta-sheet twisting relative to a "flat" beta-sheet is probably more important for structure stability in peptides and small proteins like the WW domains than in larger proteins for which there exists a significant contribution to stability arising from their extensive hydrophobic cores.     

Elongation of the BH8 β-hairpin peptide: Electrostatic interactions in β-hairpin formation and stability

An elongated version of the de novo designed beta-hairpin peptide, BH8, has allowed us to gain insight into the role of electrostatic interactions in beta-hairpin stability. A Lys-Glu electrostatic pair has been introduced by adding a residue at the beginning and at the end of the N-terminal and C-terminal strands, respectively, of the beta-hairpin structure, in both orientations. The two resulting peptides and controls having Ala residues at these positions and different combinations of Ala with Lys, or Glu residues, have been analyzed by nuclear magnetic resonance (NMR), under different pH and ionic strength conditions. All of the NMR parameters, in particular the conformational shift analysis of Calpha protons and the coupling constants, (3)J(HNalpha), correlate well and the population estimates are in reasonable agreement among the different methods used. In the most structured peptides, we find an extension of the beta-hairpin structure comprising the two extra residues. Analysis of the pH and salt dependence shows that ionic pairs contribute to beta-hairpin stability. The interaction is electrostatic in nature and can be screened by salt. There is also an important salt-independent contribution of negatively charged groups to the stability of this family of beta-hairpin peptides.     

Importance of secondary structural specificity determinants in protein folding: insertion of a native beta-sheet sequence into an alpha-helical coiled-coil

To examine how a short secondary structural element derived from a native protein folds when in a different protein environment, we inserted an 11-residue beta-sheet segment (cassette) from human immunoglobulin fold, Fab new, into an alpha-helical coiled-coil host protein (cassette holder). This de novo design protein model, the structural cassette mutagenesis (SCM) model, allows us to study protein folding principles involving both short- and long-range interactions that affect secondary structure stability and conformation. In this study, we address whether the insertion of this beta-sheet cassette into the alpha-helical coiled-coil protein would result in conformational change nucleated by the long-range tertiary stabilization of the coiled-coil, therefore overriding the local propensity of the cassette to form beta-sheet, observed in its native immunoglobulin fold. The results showed that not only did the nucleating helices of the coiled-coil on either end of the cassette fail to nucleate the beta-sheet cassette to fold with an alpha-helical conformation, but also the entire chimeric protein became a random coil. We identified two determinants in this cassette that prevented coiled-coil formation: (1) a tandem dipeptide NN motif at the N-terminal of the beta-sheet cassette, and (2) the hydrophilic Ser residue, which would be buried in the hydrophobic core if the coiled-coil structure were to fold. By amino acid substitution of these helix disruptive residues, that is, either the replacement of the NN motif with high helical propensity Ala residues or the substitution of Ser with Leu to enhance hydrophobicity, we were able to convert the random coil chimeric protein into a fully folded alpha-helical coiled-coil. We hypothesized that this NN motif is a "secondary structural specificity determinant" which is very selective for one type of secondary structure and may prevent neighboring residues from adopting an alternate protein fold. These sequences with secondary structural specificity determinants have very strong local propensity to fold into a specific secondary structure and may affect overall protein folding by acting as a folding initiation site.     

Glucosylation of beta-lactoglobulin lowers the heat capacity change of unfolding; a unique way to affect protein thermodynamics

Chemical glycosylation of proteins occurs in vivo spontaneously, especially under stress conditions, and has been linked in a number of cases to diseases related to protein denaturation and aggregation. It is the aim of this work to study the origin of the change in thermodynamic properties due to glucosylation of the folded beta-lactoglobulin A. Under mild conditions Maillard products can be formed by reaction of epsilon-amino groups of lysines with the reducing group of, in this case, glucose. The formed conjugates described here have an average degree of glycosylation of 82%. No impact of the glucosylation on the protein structure is detected, except that the Stokes radius was increased by approximately 3%. Although at ambient temperatures the change in Gibbs energy of unfolding is reduced by 20%, the denaturation temperature is increased by 5 degrees C. Using a combination of circular dichroism, fluorescence, and calorimetric approaches, it is shown that the change in heat capacity upon denaturation is reduced by 60% due to the glucosylation. Since in the denatured state the Stokes radius of the protein is not significantly smaller for the glucosylated protein, it is suggested that the nonpolar residues associate to the covalently linked sugar moiety in the unfolded state, thereby preventing their solvent exposure. In this way coupling of small reducing sugar moieties to solvent exposed groups of proteins offers an efficient and unique tool to deal with protein stability issues, relevant not only in nature but also for technological applications.     

Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia

In the mammary gland, epithelial cells are embedded in a ‘soft' environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for β-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata. We found that maintenance of β-casein expression required both laminin signalling and a ‘soft' extracellular matrix, as is the case in normal tissues in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signalling, led to the loss of β-casein expression and non-biomimetic intracellular elasticity. Our data indicate that tissue-specific gene expression is controlled by both the tissues' unique biochemical milieu and mechanical properties, processes involved in maintenance of tissue integrity and protection against tumorigenesis.     

Lipids as cofactors in protein folding: stereo-specific lipid-protein interactions are required to form HAMLET (human alpha-lactalbumin made lethal to tumor cells)

Proteins can adjust their structure and function in response to shifting environments. Functional diversity is created not only by the sequence but by changes in tertiary structure. Here we present evidence that lipid cofactors may enable otherwise unstable protein folding variants to maintain their conformation and to form novel, biologically active complexes. We have identified unsaturated C18 fatty acids in the cis conformation as the cofactors that bind apo alpha-lactalbumin and form HAMLET (human alpha-lactalbumin made lethal to tumor cells). The complexes were formed on an ion exchange column, were stable in a molten globule-like conformation, and had attained the novel biological activity. The protein-fatty acid interaction was specific, as saturated C18 fatty acids, or unsaturated C18:1trans conformers were unable to form complexes with apo alpha-lactalbumin, as were fatty acids with shorter or longer carbon chains. Unsaturated cis fatty acids other than C18:1:9cis were able to form stable complexes, but these were not active in the apoptosis assay. The results demonstrate that stereo-specific lipid-protein interactions can stabilize partially unfolded conformations and form molecular complexes with novel biological activity. The results offer a new mechanism for the functional diversity of proteins, by exploiting lipids as essential, tissue-specific cofactors in this process.     

Polycation-induced oligomerization and accelerated fibrillation of human alpha-synuclein in vitro

The aggregation and fibrillation of alpha-synuclein has been implicated as a causative factor in Parkinson's disease and several other neurodegenerative disorders known as synucleinopathies. The effect of different factors on the process of fibril formation has been intensively studied in vitro. We show here that alpha-synuclein interacts with different unstructured polycations (spermine, polylysine, polyarginine, and polyethyleneimine) to form specific complexes. In addition, the polycations catalyze alpha-synuclein oligomerization. The formation of alpha-synuclein-polycation complexes was not accompanied by significant structural changes in alpha-synuclein. However, alpha-synuclein fibrillation was dramatically accelerated in the presence of polycations. The magnitude of the accelerating effect depended on the nature of the polymer, its length, and concentration. The results illustrate the potential critical role of electrostatic interactions in protein aggregation, and the potential role of naturally occurring polycations in modulating alpha-synuclein aggregation.     

Identification of a region in G protein γ subunits conserved across species but hypervariable among subunit isoforms

The heterotrimeric GTP binding proteins, G proteins, consist of three distinct subunits: alpha, beta, and gamma. There are 12 known mammalian gamma subunit genes whose products are the smallest and most variable of the G protein subunits. Sequencing of the bovine brain gamma(10) protein by electrospray mass spectrometry revealed that it differs from the human protein by an Ala to Val substitution near the N-terminus. Comparison of gamma isoform subunit sequences indicated that they vary substantially more at the N-terminus than at other parts of the protein. Thus, species variation of this region might reflect the lack of conservation of a functionally unimportant part of the protein. Analysis of 38 gamma subunit sequences from four different species shows that the N-terminus of a given gamma subunit isoform is as conserved between different species as any other part of the protein, including highly conserved regions. These data suggest that the N-terminus of gamma is a functionally important part of the protein exhibiting substantial isoform-specific variation.