Aggregation-prone Species Research Articles

Investigation on Structural Features and Antiaggregation Properties of Chaperonins and Chaperon Like Molecules

Molecular chaperones play essential and several roles in many cellular processes, including protein folding, targeting, transport, and are essential in fighting the consequences of protein misfolding and aggregation or enhancing disaggregation of toxic aggregates by clearance mechanisms. In particular, recent studies have attributed to some chaperones the capability to directly interact with amyloid aggregation-prone species of peptide involved in Alzheimer Disease in solution and/or suppress the associated toxicity. Among them are the principal heat-shock proteins (HSPs), the small HSPs, but also molecules the exhibit marked chaperon like activity, like milk caseins or BRICHOS domains. There are several mechanisms by which chaperones exert their protective action, many of which involve protein disordered regions. However, in many cases, the scarcity of structural data has impeded an understanding of the recognition and antiaggregation mechanisms, that result crucially important when considering how to screen for and characterize potential inhibitors of amyloidosis. Here we show how a human chaperonin, Hsp60, and intrinsically disorder colloids with marked chaperon-like activity, the milk caseins, can specifically influence Aβ40 fibrillogenesis. Inhibition studies are correlated with investigation on structural, self organization and stability properties of the chaperones under study performed by a battery of biophysical methods and aimed to understand what are molecular determinants responsible for their inhibitory action.

Overcoming Heterogeneity to Study the Structure and Assembly of Small Heat-Shock Protein Chaperones with Non-Aggregating Clients

The small heat-shock proteins (sHSPs) are a family of molecular chaperones that act as a first line of defence in stress response to prevent irreversible aggregation of denaturing proteins in the cell. Most sHSPs form large oligomeric ensembles, often adopting multiple interconverting stoichiometries and architectures. This heterogeneity presents a challenge for many biophysical techniques. Native mass spectrometry (MS) is a useful method for characterising noncovalent protein assemblies, and has been applied to the sHSPs to identify oligomeric states. However, aggregation-prone species hinder MS studies, and so the effect of client binding on sHSP quaternary organisation is largely unknown. Recent interactions have been identified between sHSPs and proteins that have no known propensity to aggregate, suggesting some novel sHSP function apart from the canonical holdase chaperone. Here we examine two such interactions that occur in cardiac muscle: αB-crystallin with cardiac titin, and cvHSP with α-filamin. Using a robust selective fragmentation method to probe the full oligomeric population in subsets, we show that sHSP oligomers incorporate domains of these clients in relatively small numbers, leaving the overall oligomeric architecture intact. We further show that narrowing sHSP-client interactions to a specific sub-domain of the sHSP monomer disrupts the heterogeneity of the system, simplifying it such that the regions of interest form co-crystals that can be analysed to characterise the interactions at high resolution.

Energy landscapes of functional proteins are inherently risky.

Evolutionary pressure for protein function leads to unavoidable sampling of conformational states that are at risk of misfolding and aggregation. The resulting tension between functional requirements and the risk of misfolding and/or aggregation in the evolution of proteins is becoming more and more apparent. One outcome of this tension is sensitivity to mutation, in which only subtle changes in sequence that may be functionally advantageous can tip the delicate balance toward protein aggregation. Similarly, increasing the concentration of aggregation-prone species by reducing the ability to control protein levels or compromising protein folding capacity engenders increased risk of aggregation and disease. In this Perspective, we describe examples that epitomize the tension between protein functional energy landscapes and aggregation risk. Each case illustrates how the energy landscapes for the at-risk proteins are sculpted to enable them to perform their functions and how the risks of aggregation are minimized under cellular conditions using a variety of compensatory mechanisms.

Amyloid beta(1–42) in aqueous environments: Effects of ionic strength and E22Q (Dutch) mutation

Development of extracellular plaques characteristic of Alzheimer's disease is related to aggregation of amyloid peptides. The Aβ-42 peptide is the most aggregation prone species, and some missense mutant forms increase this aggregation ability. Due to its poor solubility as monomer in aqueous solutions, Aβ-42 conformational transitions in water have been largely investigated by molecular dynamics. Here we report an all-atom molecular dynamics analysis of the Aβ-42 peptide in aqueous environment using as starting conformation a structure obtained in an isotropic, low-polarity medium, representing a plausible model for the membrane-bound species. While previous studies commonly show that Aβ-42 is largely unstructured in aqueous solution, here we report that this peptide can adopt partially folded structures. Importance of ionic strength has been also investigated, showing that at physiological ionic strength condition a loop stabilizing electrostatic interaction involving Lys28 builds up. In addition, besides stable α-helix structures, we observe the appearance of 310 helix, similar to what was reported experimentally for the Aβ-40 species. The effect of E22Q (Dutch) mutation in high ionic strength condition has been explored. We show that this mutation has a dramatic impact on the Aβ-42 structure. Instead of a partially folded, but extended, conformation obtained with the wild type, the E22Q assumes a two-helix collapsed one due to the clustering of hydrophobic residues.

Truncation of a β-Barrel Scaffold Dissociates Intrinsic Stability from Its Propensity to Aggregation

Δ98Δ is a functional all-β sheet variant of intestinal fatty acid binding protein (IFABP) that was generated by controlled proteolysis. This framework is useful to study the molecular determinants related to aggregation of β-barrel proteins. Albeit displaying increased conformational plasticity, Δ98Δ exhibits a nativelike β-barrel topology and is able to support a cooperative folding behavior. Here we present a comparative study of IFABP and Δ98Δ regarding their conformational perturbation and aggregation propensity triggered by trifluoroethanol. Both proteins share a common nucleation-elongation mechanism, whereby the rate-limiting step is the formation of stable dimeric nuclei followed by the association of monomers to the growing aggregates. Despite leading to a less stable structure, the extensive truncation of IFABP yields a form exhibiting a somewhat lower tendency to aggregate. This finding appears at odds with the established notion that a perturbation of the native compact fold should necessarily favor the population of aggregation-prone species. In addition to the aggregation propensity dictated by a given amino-acid sequence, our contention holds that long-range interactions might also play a major role in determining the overall aggregation propensity.

Phenylpiperidine-type γ-secretase modulators target the transmembrane domain 1 of presenilin 1

Amyloid-β peptide ending at the 42nd residue (Aβ42) is implicated in the pathogenesis of Alzheimer's disease (AD). Small compounds that exhibit selective lowering effects on Aβ42 production are termed γ-secretase modulators (GSMs) and are deemed as promising therapeutic agents against AD, although the molecular target as well as the mechanism of action remains controversial. Here, we show that a phenylpiperidine-type compound GSM-1 directly targets the transmembrane domain (TMD) 1 of presenilin 1 (PS1) by photoaffinity labelling experiments combined with limited digestion. Binding of GSM-1 affected the structure of the initial substrate binding and the catalytic sites of the γ-secretase, thereby decreasing production of Aβ42, possibly by enhancing its conversion to Aβ38. These data indicate an allosteric action of GSM-1 by directly binding to the TMD1 of PS1, pinpointing the target structure of the phenylpiperidine-type GSMs.

Beta-Sheet Features in the Greek Key Gamma D-Crystallin Recognized by the Lens Chaperone Alpha B-Crystallin

The γ-crystallins are long-lived proteins of the vertebrate lens consisting of four Greek-key motifs organized into two homologous domains. Human γD-Crys can refold in vitro to its native state after unfolding in high concentrations of GdnHCl. However, in buffer or at very low denaturant concentrations aggregation of refolding HγD-Crys intermediates competes with productive refolding. α-crystallin, a small heat shock protein chaperone, is a polydisperse complex of ∼ 800 kD consisting of homologous ∼20 kD αA- and αB-crystallin chains (αA- and αB-Crys). Its chaperone activity involves suppressing aggregation by binding aggregation-prone species. Aggregates isolated from mature-onset cataracts, the major cause of blindness worldwide, contain damaged and misfolded forms of βγ-crystallins. We had previously determined that the conformation of the bound HγD-Crys substrate in γD_αB complexes resembles the partially folded intermediate populated during refolding/unfolding equilibrium experiments. The chaperone/substrate interaction have been probed further using single domain constructs. The HγD-Crys C-terminal domain construct (γD-Ctd) aggregated upon refolding, while the N-terminal domain construct (γD-Ntd) did not aggregate significantly under similar conditions. HαB-Crys suppressed the aggregation of the γD-Ctd and formed long-lived γD-Ctd_αB complexes. Using a double mutant of HαB-Crys (W9F/W60F) lacking tryptophans, we have determined, through the fluorescence emission of substrate γD-Ctd tryptophans, that the γD-Ctd in the γD-Ctd_αB complexes is partially unfolded. The results are consistent with models in which the tryptophans normally buried in the Greek key, interact with the chaperone in the complex.

Metal-Free ALS Variants of Dimeric Human Cu,Zn-Superoxide Dismutase Have Enhanced Populations of Monomeric Species

Amino acid replacements at dozens of positions in the dimeric protein human, Cu,Zn superoxide dismutase (SOD1) can cause amyotrophic lateral sclerosis (ALS). Although it has long been hypothesized that these mutations might enhance the populations of marginally-stable aggregation-prone species responsible for cellular toxicity, there has been little quantitative evidence to support this notion. Perturbations of the folding free energy landscapes of metal-free versions of five ALS-inducing variants, A4V, L38V, G93A, L106V and S134N SOD1, were determined with a global analysis of kinetic and thermodynamic folding data for dimeric and stable monomeric versions of these variants. Utilizing this global analysis approach, the perturbations on the global stability in response to mutation can be partitioned between the monomer folding and association steps, and the effects of mutation on the populations of the folded and unfolded monomeric states can be determined. The 2- to 10-fold increase in the population of the folded monomeric state for A4V, L38V and L106V and the 80- to 480-fold increase in the population of the unfolded monomeric states for all but S134N would dramatically increase their propensity for aggregation through high-order nucleation reactions. The wild-type-like populations of these states for the metal-binding region S134N variant suggest that even wild-type SOD1 may also be prone to aggregation in the absence of metals.

Characterization of the small heat shock protein αB‐crystallin chaperone binding regions in human γD‐crystallin using single domain constructs of γD‐crystallin

α‐crystallin, an ATP‐independent small heat shock protein, is one of the ubiquitous crystallins in vertebrate lenses. It is a polydisperse complex of ~ 800 kD consisting of two subunits (~20 kD) αA‐ and αB‐crystallin (αA‐ and αB‐Crys). Its chaperone activity involves suppressing aggregation by binding aggregation‐prone species. γD‐crystallin (γD‐Crys), a member of the βγ‐crystallin family, is localized in the lens nucleus. Aggregates isolated from mature‐onset cataracts contain damaged and misfolded forms of γD‐Crys. Human γD‐Crys refolds through the population of a stable, partially folded intermediate in vitro, but aggregation reactions compete with productive folding. Our previous work showed that HγD‐Crys substrate in γD—αB complexes resembles this folding intermediate, which has its N‐terminal domain unfolded and its C‐terminal domain folded. Using single domain constructs of HγD‐Crys, we showed that the C‐terminal domain construct (γD‐Ctd) aggregates upon refolding, while the N‐terminal domain construct (γD‐Ntd) does not aggregate under similar conditions. Unfolding and refolding γD‐Ctd and γD‐Ntd together at equal molar ratios resulted in γD‐Ctd recruiting γD‐Ntd into the aggregate. HαB‐Crys can suppress the aggregation of the γD‐Ctd and forms γD‐Ctd—αB complexes. These results provide further insight into how α‐crystallin interacts with aggregation‐prone substrates.

Chaperone Interactions of the Small Heat Shock Protein Human αb-Crystallin With Its Physiological Substrate γd-Crystallin and Its Isolated Domains

α-crystallin, a small heat shock protein chaperone, is one of the ubiquitous crystallins in the vertebrate lens, along with the βγ-crystallins. It is a polydisperse complex of ∼800 kD consisting of two subunits (∼20 kD) αA- and αB-crystallin (αA- and αB-Crys). Its chaperone activity involves suppressing aggregation by binding aggregation-prone species. Aggregates isolated from mature-onset cataracts, the major cause of blindness worldwide, contain damaged and misfolded forms of βγ-crystallins. The γ-crystallins are structural, monomeric proteins that consist of four Greek-key motifs organized into two domains. Human γD-crystallin (HγD-Crys) is a stable and long-lived mammalian γ-crystallin localized in the lens nucleus. It can refold in vitro to its native state after unfolding in high concentrations of GdnHCl. However, at very low denaturant concentrations (< 1 M GdnHCl) aggregation of refolding HγD-Crys intermediates competes with productive refolding. We have previously determined that the conformation of the bound HγD-Crys substrate in γD-αB complexes resembles the partially folded intermediate populated during refolding/unfolding equilibrium experiments, which has its N-terminal domain unfolded and its C-terminal domain folded. We have utilized single domain constructs to further characterize the binding interactions of HαB-Crys to different regions of HγD-Crys. The HγD-Crys C-terminal domain construct (γD-Ctd) aggregates upon refolding, while the N-terminal domain construct (γD-Ntd) does not aggregate under similar conditions. However, when γD-Ctd and γD-Ntd are unfolded and refolded together, γD-Ctd recruits γD-Ntd into the aggregate. HαB-Crys can suppress the aggregation of the γD-Ctd and forms γD-Ctd-αB complexes. Using a no-Trp mutant of HαB-Crys (W9F/W60F), we have determined, through the fluorescence emission of γD-Ctd tryptophans, that the γD-Ctd in the γD-Ctd-αB complexes is partially folded. These results provide further insight into how α-crystallin interacts with aggregation-prone substrates in vivo.

Human αB‐Crystallin suppresses the aggregation in vitro of partially folded intermediates of human γC‐, γD‐, and γS‐Crystallins

α‐crystallin, a small heat shock protein chaperone, is one of the ubiquitous crystallins in the vertebrate lens, along with the ??‐crystallins. It is a polydisperse complex of ~ 800 kD consisting of two subunits (~20 kD) αA‐ and αB‐crystallin (αA‐ and αB‐crys). Its chaperone activity involves suppressing aggregation by binding aggregation‐prone species. Aggregates isolated from mature‐onset cataracts, the major cause of blindness worldwide, contain damaged and misfolded forms of ??‐crystallins. Human ?D‐ and ?C‐crystallin (H?D‐ and H?C‐crys) are stable, long‐lived ?‐crystallins in the lens nucleus, while ?S‐crystallin (H?S‐crys) is abundant in the lens cortex. All three ?‐crystallins can refold in vitro to their native state after unfolding in high concentrations of GdnHCl. Refolding unfolded H?C‐, H?D‐, or H?S‐crys to very low GdnHCl concentrations (&lt; 1 M) resulted in partially folded intermediates partitioning between productive refolding and aggregation pathways. H?D‐, H?C‐ or H?S‐Crys protein was allowed to refold and aggregate in the presence of HαB‐Crys at different monomer‐to‐monomer ratios. H?D‐ and H?C‐Crys aggregation was suppressed to similar levels, whereas H?S‐Crys aggregation was not suppressed as well under identical conditions in assays measuring solution turbidity at 350 nm. These results provide a model for how α‐crystallin interacts with aggregation‐prone substrates in vivo

Amyloidogenic potential of α‐chymotrypsin in different conformational states

Amyloid fibril formation is widely believed to be a generic property of polypeptide chains. In the present study, alpha-chymotrypsin, a well-known serine protease has been driven toward these structures by the use of two different conditions involving (I) high temperature, pH 2.5, and (II) low concentration of trifluoroethanol (TFE), pH 2.5. A variety of experimental methods, including fluorescence emission, dynamic quenching, steady-state fluorescence anisotropy, far-UV circular dichroism, nuclear magnetic resonance spectroscopy, and dynamic light scattering were employed to characterize the conformational states of alpha-chymotrypsin that precede formation of amyloid fibrils. The structure formed under Condition I was an unfolded monomer, whereas an alpha-helical rich oligomer was induced in Condition II. Both the amyloid aggregation-prone species manifested a higher solvent exposure of hydrophobic and aromatic residues compared with the native state. Upon incubation of the protein in these conditions for 48 h, amyloid-like fibrils were formed with diameters of about 10-12 nm. In contrast, at neutral pH and low concentration of TFE, a significant degree of amorphous aggregation was observed, suggesting that charge neutralization of acidic residues in the amyloid core region has a positive influence on amyloid fibril formation. In summary, results presented in this communication suggest that amyloid fibrils of alpha-chymotrypsin may be obtained from a variety of structurally distinct conformational ensembles highlighting the critical importance of protein evolution mechanisms related to prevention of protein misfolding.

Hydrophilic Residues 526KNDAAD531 in the Flexible C-terminal Region of the Chaperonin GroEL Are Critical for Substrate Protein Folding within the Central Cavity

The final 23 residues in the C-terminal region of Escherichia coli GroEL are invisible in crystallographic analyses due to high flexibility. To probe the functional role of these residues in the chaperonin mechanism, we generated and characterized C-terminal truncated, double ring, and single ring mutants of GroEL. The ability to assist the refolding of substrate proteins rhodanese and malate dehydrogenase decreased suddenly when 23 amino acids were truncated, indicating that a sudden change in the environment within the central cavity had occurred. From further experiments and analyses of the hydropathy of the C-terminal region, we focused on the hydrophilicity of the sequence region (26 KNDAAD 531 and generated two GroEL mutants where these residues were changed to a neutral hydropathy sequence (526 GGGAAG 531) and a hydrophobic sequence (526 IGIAAI 531), respectively. Very interestingly, the two mutants were found to be defective in function both in vitro and in vivo. Deterioration of function was not observed in mutants where this region was replaced by a scrambled (526 NKADDA 531) or homologous (526 RQEGGE 531) sequence, indicating that the hydrophilicity of this sequence was important. These results highlight the importance of the hydrophilic nature of 526 KNDAAD 531 residues in the flexible C-terminal region for proper protein folding within the central cavity of GroEL.

Cat and Mouse

The role of cathepsin B, a lysosomal protease implicated in amyloid-beta (Abeta1-42) metabolism, in Alzheimer's disease remains controversial. In this issue of Neuron, Mueller-Steiner et al. manipulate the expression of cathepsin B in aged APP transgenic mice, observing that increased expression degrades preformed oligomeric and fibrillar amyloid, while inactivation accelerates beta-amyloidosis.

Antiamyloidogenic and Neuroprotective Functions of Cathepsin B: Implications for Alzheimer's Disease

Alzheimer's disease (AD) may result from the accumulation of amyloid-beta (Abeta) peptides in the brain. The cysteine protease cathepsin B (CatB) is associated with amyloid plaques in AD brains and has been suspected to increase Abeta production. Here, we demonstrate that CatB actually reduces levels of Abeta peptides, especially the aggregation-prone species Abeta1-42, through proteolytic cleavage. Genetic inactivation of CatB in mice with neuronal expression of familial AD-mutant human amyloid precursor protein (hAPP) increased the relative abundance of Abeta1-42, worsening plaque deposition and other AD-related pathologies. Lentivirus-mediated expression of CatB in aged hAPP mice reduced preexisting amyloid deposits, even thioflavin S-positive plaques. Under cell-free conditions, CatB effectively cleaved Abeta1-42, generating C-terminally truncated Abeta peptides that are less amyloidogenic. Thus, CatB likely fulfills antiamyloidogenic and neuroprotective functions. Insufficient CatB activity might promote AD; increasing CatB activity could counteract the neuropathology of this disease.

Protein Structural Conformation and Not Second Virial Coefficient Relates to Long-Term Irreversible Aggregation of a Monoclonal Antibody and Ovalbumin in Solution

The purpose of the study was to investigate the relationship of the second virial coefficient, B22, to the extent of irreversible protein aggregation upon storage. A monoclonal antibody and ovalbumin were incubated at 37 degrees C (3 months) under various solution conditions to monitor the extent of aggregation. The B22 values of these proteins were determined under similar solution conditions by a modified method of flow-mode static light scattering. The conformation of these proteins was studied using circular dichroism (CD) spectroscopy and second-derivative Fourier transform infrared spectroscopy. Both proteins readily aggregated at pH 4.0 (no aggregation observed at pH 7.4); the extent of aggregation varied with the ionic strength and the presence of cosolutes (sucrose, glycine, and Tween 80). Debye plots of the monoclonal antibody showed moderate attractive interactions at pH 7.4, whereas, at pH 4.0, nonlinear plots were obtained, indicating self-association. CD studies showed partially unfolded structure of antibody at pH 4.0 compared with that at pH 7.4. In the case of ovalbumin, similar B22 values were obtained in all solution conditions irrespective of whether the protein aggregated or not. CD studies of ovalbumin indicated the presence of a fraction of completely unfolded as well as partially unfolded species at pH 4.0 compared with that at pH 7.4. The formation of a structurally altered state is a must for irreversible aggregation to proceed. Because this aggregation-prone species could be an unfolded species present in a small fraction compared with that of the native state or it could be a partially unfolded state whose net interactions are not significantly different compared with those of the native state, yet the structural changes are sufficient to lead to long-term aggregation, it is unlikely that B22 will correlate with long-term aggregation.

Polyvinylpyrrolidone 40 Assists the Refolding of Bovine Carbonic Anhydrase B by Accelerating the Refolding of the First Molten Globule Intermediate

Protecting proteins from aggregation is one of the most important issues in both protein science and protein engineering. In this research, the mechanism of enhancing the refolding of guanidine hydrochloride-denatured carbonic anhydrase B by polyvinylpyrrolidone 40 (PVP40) was studied by both kinetic and equilibrium refolding experiments. The reactivation and refolding kinetics indicated that the rate constant of refolding the first refolding intermediate (I(1)) to the second one (I(2)) is promoted by the addition of PVP. Fluorescence quenching studies further indicated that PVP could bind to the aggregation-prone species I(1), resulting in the protection of the exposed hydrophobic surface, a minimization of the protein surface, and more importantly, an increase of the refolding rate of I(1). These properties were quite different from those of poly(ethylene glycol) (PEG), which has been shown to have a strong and stoichiometric binding to I(1) and does not interfere with the refolding pathway. Unlike PEG, the binding of PVP to I(1) does not block the aggregation pathway directly but decreases the energy barrier for I(1) to refold to I(2) and thus reduces the accumulation of I(1). These results suggested that PVP works by a quite different mechanism from those well established ones in chaperones and chemical promoters. PVP is more like a folding catalyst rather than a chemical chaperone. The distinct mechanism of enhancing protein aggregation by PVP is expected to facilitate the attempt to develop new chemical compounds as well as new strategies to protect proteins from aggregation.

Amyloid formation under physiological conditions proceeds via a native-like folding intermediate

Although most proteins can assemble into amyloid-like fibrils in vitro under extreme conditions, how proteins form amyloid fibrils in vivo remains unresolved. Identifying rare aggregation-prone species under physiologically relevant conditions and defining their structural properties is therefore an important challenge. By solving the folding mechanism of the naturally amyloidogenic protein beta-2-microglobulin at pH 7.0 and 37 degrees C and correlating the concentrations of different species with the rate of fibril elongation, we identify a specific folding intermediate, containing a non-native trans-proline isomer, as the direct precursor of fibril elongation. Structural analysis using NMR shows that this species is highly native-like but contains perturbation of the edge strands that normally protect beta-sandwich proteins from self-association. The results demonstrate that aggregation pathways can involve self-assembly of highly native-like folding intermediates, and have implications for the prevention of this, and other, amyloid disorders.

Unscrambling an egg: protein disaggregation by AAA+ proteins

A protein quality control system, consisting of molecular chaperones and proteases, controls the folding status of proteins and prevents the aggregation of misfolded proteins by either refolding or degrading aggregation-prone species. During severe stress conditions this protection system can be overwhelmed by high substrate load, resulting in the formation of protein aggregates. In such emergency situations, Hsp104/ClpB becomes a key player for cell survival, as it has the extraordinary capacity to rescue proteins from an aggregated state in cooperation with an Hsp70 chaperone system. The ring-forming Hsp104/ClpB chaperone belongs to the AAA+ protein superfamily, which in general drives the assembly and disassembly of protein complexes by ATP-dependent remodelling of protein substrates. A disaggregation activity was also recently attributed to other eubacterial AAA+ proteins, while such an activity has not yet been identified in mammalian cells. In this review, we report on new insights into the mechanism of protein disaggregation by AAA+ proteins, suggesting that these chaperones act as molecular crowbars or ratchets.

Insight into the Secondary Structure of Non-native Proteins Bound to a Molecular Chaperone α-Crystallin: AN ISOTOPE-EDITED INFRARED SPECTROSCOPIC STUDY

alpha-Crystallin, the major lens protein, acts as a molecular chaperone by preventing the aggregation of proteins damaged by heat and other stress conditions. To characterize the backbone conformation of protein folding intermediates that are recognized by the chaperone, we prepared the uniformly (13)C-labeled alphaA-crystallin. The labeling greatly reduced the overlapping between the conformation-sensitive amide I bands of alpha-crystallin and unlabeled substrate proteins. This procedure has allowed us to gain insight into the secondary structure of alpha-crystallin-bound species, an understanding which has previously been unattainable. Analysis of the infrared spectra of two substrate proteins (gamma- and beta(L)-crystallins) indicates that heat-destabilized conformers captured by alpha-crystallin are characterized by a high proportion of native-like secondary structure. In contrast to the chaperone-bound species, the same proteins subjected to heat treatment in the absence of alpha-crystallin preserve very little native secondary structure. These data show that alpha-crystallin specifically recognizes very early intermediates on the denaturation pathway of proteins. These aggregation-prone species are characterized by native-like secondary structure but compromised tertiary interactions. The experimental approach described in this study can be further applied to probe the backbone conformation of proteins bound to chaperones other than alpha-crystallin.