Low Denaturant Concentrations Research Articles

Conservation of kinetic stability, but not the unfolding mechanism, between human transthyretin and a transthyretin-related enzyme

Kinetic stability is thought to be an attribute of proteins that require a long lifetime, such as the transporter of thyroxine and holo retinol-binding protein or transthyretin (TTR) functioning in the bloodstream, cerebrospinal fluid, and vitreous humor. TTR evolved from ancestral enzymes known as TTR-related proteins (TRPs). Here, we develop a rate-expansion approach that allows unfolding rates to be measured directly at low denaturant concentration, revealing that kinetic stability exists in the Escherichia coli TRP (EcTRP), even though the enzyme structure is more energetically frustrated and has a more mutation-sensitive folding mechanism than human TTR. Thus, the ancient tetrameric enzyme may already have been poised to mutate into a kinetically stable human transporter. An extensive mutational study that exchanges residues at key sites within the TTR and EcTRP dimer-dimer interface shows that tyrosine 111, replaced by a threonine in TTR, is the gatekeeper of frustration in EcTRP because it is critical for function. Frustration, virtually absent in TTR, occurs at multiple sites in EcTRP and even cooperatively for certain pairs of mutations. We present evidence that evolution at the C terminus of TTR was a compensatory event to maintain the preexisting kinetic stability while reducing frustration and sensitivity to mutation. We propose an "overcompensation" pathway from EcTRPs to functional hybrids to modern TTRs that is consistent with the biophysics discussed here. An alternative plausible pathway is also presented.

Influence of denaturants on amyloid β42 aggregation kinetics.

Amyloid formation is linked to devastating neurodegenerative diseases, motivating detailed studies of the mechanisms of amyloid formation. For Aβ, the peptide associated with Alzheimer’s disease, the mechanism and rate of aggregation have been established for a range of variants and conditions in vitro and in bodily fluids. A key outstanding question is how the relative stabilities of monomers, fibrils and intermediates affect each step in the fibril formation process. By monitoring the kinetics of aggregation of Aβ42, in the presence of urea or guanidinium hydrochloride (GuHCl), we here determine the rates of the underlying microscopic steps and establish the importance of changes in relative stability induced by the presence of denaturant for each individual step. Denaturants shift the equilibrium towards the unfolded state of each species. We find that a non-ionic denaturant, urea, reduces the overall aggregation rate, and that the effect on nucleation is stronger than the effect on elongation. Urea reduces the rate of secondary nucleation by decreasing the coverage of fibril surfaces and the rate of nucleus formation. It also reduces the rate of primary nucleation, increasing its reaction order. The ionic denaturant, GuHCl, accelerates the aggregation at low denaturant concentrations and decelerates the aggregation at high denaturant concentrations. Below approximately 0.25 M GuHCl, the screening of repulsive electrostatic interactions between peptides by the charged denaturant dominates, leading to an increased aggregation rate. At higher GuHCl concentrations, the electrostatic repulsion is completely screened, and the denaturing effect dominates. The results illustrate how the differential effects of denaturants on stability of monomer, oligomer and fibril translate to differential effects on microscopic steps, with the rate of nucleation being most strongly reduced.

Stability of proteins encapsulated in Michael-type addition polyethylene glycol hydrogels.

Degradable polyethylene glycol (PEG) hydrogels are excellent vehicles for sustained drug release due to their biocompatibility, tunable physical properties, and customizable degradation. However, protein therapeutics are unstable under physiological conditions and releasing degraded or inactive therapeutics can induce immunogenic effects. While controlling protein release from PEG hydrogels has been extensively investigated, few studies have detailed protein stability long-term or under stress conditions. Here, lysozyme and alcohol dehydrogenase (ADH) stability were explored upon encapsulation in PEG hydrogels formed through Michael-type addition. The stability and structure of the two model proteins were monitored by measuring the free energy of unfolding and fluoresce quenching when confined in a hydrogel and compared to PEG solution and buffer. Hydrogels destabilized lysozyme structure at low denaturant concentrations but prevented complete unfolding at high concentrations. ADH was stabilized as the confining mesh size approached the protein radius of gyration. Both proteins retained enzymatic activity within the hydrogels under stress conditions, including denaturant, high temperature, and agitation. Conjugation between lysozyme and PEG-acrylate was identified at long reaction times but no conjugation was observed in the time required for complete gelation. Studies of protein stability in PEG hydrogels, as the one detailed here, can lead to designer technologies for the improved formulation, storage, and delivery of protein therapeutics.

Structurally distinct unfolding intermediates formed from a staphylococcal capsule-producing enzyme retained NADPH binding activity

CapF, a capsule-producing enzyme expressed by Staphylococcus aureus, binds NADPH and exists as a dimer in the aqueous solution. Many other capsule-producing virulent bacteria also express CapF orthologs. To understand the folding-unfolding mechanism of S. aureus CapF, herein a recombinant CapF (rCapF) was individually investigated using urea and guanidine hydrochloride (GdnCl). Unfolding of rCapF by both the denaturants was reversible but proceeded via the synthesis of a different number of intermediates. While two dimeric intermediates (rCapF4 and rCapF5) were formed at 0.5 M and 1.5 M GdnCl, three dimeric intermediates (rCapF1, rCapF2, and rCapF3) were produced at 1 M, 2 M, and 3 M urea, respectively. rCapF5 showed 3.6 fold less NADPH binding activity, whereas other intermediates retained full NADPH binding activity. Compared to rCapF, all of the intermediates (except rCapF3) had a compressed shape. Conversely, rCapF3 possessed a native protein-like shape. The maximum shape loss was in rCapF4 though its secondary structure remained unperturbed. Additionally, the tertiary structure and hydrophobic surface area of the intermediates neither matched with each other nor with those of the native rCapF. Of the four Trp residues in rCapF, one or more Trp residues in the intermediates may have higher solvent accessibility. Using sequence alignment and a tertiary structural model of CapF, we have demonstrated that the region around Trp 137 of CapF may be most sensitive to unfolding, whereas the NADPH binding motif carrying region at the N-terminal end of this protein may be resistant to unfolding, particularly at the low denaturant concentrations. Communicated by Ramaswamy H. Sarma

Kinetic Stability of Long-Lived Human Lens γ-Crystallins and Their Isolated Double Greek Key Domains

The γ-crystallins of the eye lens nucleus are among the longest-lived proteins in the human body. Synthesized in utero, they must remain folded and soluble throughout adulthood to maintain lens transparency and avoid cataracts. γD- and γS-crystallin are two major monomeric crystallins of the human lens. γD-crystallin is concentrated in the oldest lens fiber cells, the lens nucleus, whereas γS-crystallin is concentrated in the younger cells of the lens cortex. The kinetic stability parameters of these two-domain proteins and their isolated domains were determined and compared. Kinetic unfolding experiments monitored by fluorescence spectroscopy in varying concentrations of guanidinium chloride were used to extrapolate unfolding rate constants and half-lives of the crystallins in the absence of the denaturant. Consistent with their long lifespans in the lens, extrapolated half-lives for the initial unfolding step were on the timescale of years. Both proteins’ isolated N-terminal domains were less kinetically stable than their respective C-terminal domains at denaturant concentrations predicted to disrupt the domain interface, but at low denaturant concentrations, the relative kinetic stabilities were reversed. Cataract-associated aggregation has been shown to proceed from partially unfolded intermediates in these proteins; their extreme kinetic stability likely evolved to protect the lens from the initiation of aggregation reactions. Our findings indicate that the domain interface is the source of significant kinetic stability. The gene duplication and fusion event that produced the modern two-domain architecture of vertebrate lens crystallins may be the origin of their high kinetic as well as thermodynamic stability.

Factors defining the effects of macromolecular crowding on dynamics and thermodynamic stability of heme proteins in-vitro

The role of crowding agents on structure and activities of heme proteins has been established. Analysis of kinetic and thermodynamic parameters measured for CO-dissociation reaction of natively-folded carbonmonoxycytochrome c (NCO) and carbonmonoxymyoglobin (MbCO) at different [GdnHCl] or [Urea] in the presence of crowding agents (dextran 40, dextran 70 and ficoll 70) demonstrate that (i) at low denaturant concentrations, crowder presence enhances the denaturant-mediated restricted dynamics of NCO and MbCO, and (ii) at higher denaturant concentrations, large scale unfolding-fluctuations dominate the dynamics and inclusion of crowder counteracts the structural-fluctuations causing the unfolding of proteins. Thermodynamic analysis of thermal and urea-unfolding curves of cytochrome c (Cyt c) and myoglobin (Mb) measured at different [GdnHCl] in presence of crowding agents reveals that crowder presence counterbalances and strengthens the destabilizing action of GdnHCl on stability of Cyt c and Mb, respectively. This study further demonstrates that the size, shape and concentration of crowding agent modulate the effect of crowder on denaturant-mediated dynamics and thermodynamic stability of heme proteins.

FRET studies of various conformational states adopted by transthyretin.

Transthyretin (TTR) is an extracellular protein able to deposit into well-defined protein aggregates called amyloid, in pathological conditions known as senile systemic amyloidosis, familial amyloid polyneuropathy, familial amyloid cardiomyopathy and leptomeningeal amyloidosis. At least three distinct partially folded states have been described for TTR, including the widely studied amyloidogenic state at mildly acidic pH. Here, we have used fluorescence resonance energy transfer (FRET) experiments in a monomeric variant of TTR (M-TTR) and in its W41F and W79F mutants, taking advantage of the presence of a unique, solvent-exposed, cysteine residue at position 10, that we have labelled with a coumarin derivative (DACM, acceptor), and of the two natural tryptophan residues at positions 41 and 79 (donors). Trp41 is located in an ideal position as it is one of the residues of β-strand C, whose degree of unfolding is debated. We found that the amyloidogenic state at low pH has the same FRET efficiency as the folded state at neutral pH in both M-TTR and W79F-M-TTR, indicating an unmodified Cys10-Trp41 distance. The partially folded state populated at low denaturant concentrations also has a similar FRET efficiency, but other spectroscopic probes indicate that it is distinct from the amyloidogenic state at acidic pH. By contrast, the off-pathway state accumulating transiently during refolding has a higher FRET efficiency, indicating non-native interactions that reduce the Cys10-Trp41 spatial distance, revealing a third distinct conformational state. Overall, our results clarify a negligible degree of unfolding of β-strand C in the formation of the amyloidogenic state and establish the concept that TTR is a highly plastic protein able to populate at least three distinct conformational states.

Protein collapse is encoded in the folded state architecture.

Folded states of single domain globular proteins are compact with high packing density. The radius of gyration, Rg, of both the folded and unfolded states increase as Nν where N is the number of amino acids in the protein. The values of the Flory exponent ν are, respectively, ≈⅓ and ≈0.6 in the folded and unfolded states, coinciding with those for homopolymers. However, the extent of compaction of the unfolded state of a protein under low denaturant concentration (collapsibility), conditions favoring the formation of the folded state, is unknown. We develop a theory that uses the contact map of proteins as input to quantitatively assess collapsibility of proteins. Although collapsibility is universal, the propensity to be compact depends on the protein architecture. Application of the theory to over two thousand proteins shows that collapsibility depends not only on N but also on the contact map reflecting the native structure. A major prediction of the theory is that β-sheet proteins are far more collapsible than structures dominated by α-helices. The theory and the accompanying simulations, validating the theoretical predictions, provide insights into the differing conclusions reached using different experimental probes assessing the extent of compaction of proteins. By calculating the criterion for collapsibility as a function of protein length we provide quantitative insights into the reasons why single domain proteins are small and the physical reasons for the origin of multi-domain proteins. Collapsibility of non-coding RNA molecules is similar β-sheet proteins structures adding support to "Compactness Selection Hypothesis".

Different States of Acrylodan-Labeled 3'5'-Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits in Denaturant Solutions.

Fluorescence spectroscopy was used to differentiate between different states of acrylodan-labeled cAMP-dependent protein kinase catalytic subunits in urea, guanidine hydrochloride and 3-(N-morpholino)propanesulfonic acid solutions, by measuring changes in the emission spectrum of the protein-coupled dye, which is very sensitive to its microenvironment. Decomposition of the observed fluorescence spectra by a parameterized log-normal distribution function allowed the resolution of overlapping spectral bands and revealed the formation of three distinct protein states, denominated as native, denatured and unfolded structures. At low denaturant concentrations the formation of the denatured form from the native protein was observed, and this process was characterized by a blue-shift of the fluorescence spectrum of acrylodan, indicating that the dye was transferred into some water-deficit hydrophobic environment inside the protein molecule. Therefore, formation of a "dry molten globule" structure could be suggested in state. At high denaturant concentrations a red-shift of the emission spectrum of the protein-coupled probe was observed indicating significant extrusion of the dye molecule into water environment as a result of the unfolding of the protein structure.

Probing the Molecular Origin of Native-State Flexibility in Repeat Proteins.

In contrast to globular proteins, the structure of repeat proteins is dominated by a regular set of short-range interactions. This property may confer on the native state of such proteins significant elasticity. We probe the molecular origin of the spring-like behavior of repeat proteins using a designed tetratricopeptide repeat protein with three repeat units (CTPR3). Single-molecule fluorescence studies of variants of the protein with FRET pairs at different positions show a continuous expansion of the folded state of CTPR3 at low concentrations of a chemical denaturant, preceding the all-or-none transition to the unfolded state. This remarkable native-state expansion can be explained quantitatively by a reduction in the spring constant of the structure. Circular dichroism and tryptophan fluorescence spectroscopy further show that the expansion does not involve either unwinding of CTPR3 helices or unraveling of interactions within repeats. These findings point to hydrophobic inter-repeat contacts as the source of the elasticity of repeat proteins.

Collapse of a long axis: single-molecule Förster resonance energy transfer and serpin equilibrium unfolding.

The energy required for mechanical inhibition of target proteases is stored in the native structure of inhibitory serpins and accessed by serpin structural remodeling. The overall serpin fold is ellipsoidal with one long and two short axes. Most of the structural remodeling required for function occurs along the long axis, while expansion of the short axes is associated with misfolded, inactive forms. This suggests that ellipticity, as typified by the long axis, may be important for both function and folding. Placement of donor and acceptor fluorophores approximately along the long axis or one of the short axes allows single-pair Förster resonance energy transfer (spFRET) to report on both unfolding transitions and the time-averaged shape of different conformations. Equilibrium unfolding and refolding studies of the well-characterized inhibitory serpin α1-antitrypsin reveal that the long axis collapses in the folding intermediates while the monitored short axis expands. These energetically distinct intermediates are thus more spherical than the native state. Our spFRET studies agree with other equilibrium unfolding studies that found that the region around one of the β strands, s5A, which helps define the long axis and must move for functionally required loop insertion, unfolds at low denaturant concentrations. This supports a connection between functionally important structural lability and unfolding in the inhibitory serpins.

Abstract 586: Ordered Opening of the 4-Helix Bundle in the LDL-Receptor Binding Domain of Human Apolipoprotein E3 as Revealed by Fluorescence Polarization Analysis

Apolipoprotein E3 (apoE3) is an anti-atherogenic protein that helps maintain triglycerides and cholesterol levels in the plasma. It is responsible for binding and cellular uptake of plasma lipoproteins via the low-density lipoprotein receptor (LDLr) family of proteins. It is a highly alpha-helical protein that can exist in lipid-free and lipid-bound states, and undergoes a dramatic conformational re-organization when transitioning between the two states. The objective of this study is to understand the mechanism of chemical-induced unfolding of the 4-helix bundle N-terminal (NT) domain housing the LDLr binding sites. The rationale is that the ease of unfolding is a reflection of ease of helix bundle opening during lipid interaction. We tested the hypothesis that the 4-helix bundle undergoes a concerted opening to reveal the hydrophobic interior, analogous to opening an umbrella. Using X-ray and NMR structure guided rational approach, single cysteine-containing constructs were designed; a fluorescent probe was attached to the free -SH groups to serve as a spectroscopic reporter about the microenvironment and mobility of selected segments of the protein: the 4 main helices observed in the X-ray structure (H1 to H4) and the relatively flexible helixes flanking the helix bundle as observed in the NMR structure. We induced unfolding by chemical denaturation and monitored changes in protein secondary structure and tertiary conformation by circular dichroism and fluorescence spectroscopy, respectively. The overall global was relatively unaltered in the labeled variants; however, fluorescence emission and polarization studies revealed that during unfolding, the microenvironment of the probe on H3 and H4 is more polar and that these helices become more mobile at lower denaturant concentrations, compared to probes on H1 and H2. The data suggest that the 4-helix bundle undergoes a sequential unfolding pattern of the main helices in the following order: helix 4/helix 3, helix 2 and lastly helix 1. Our results offer insights into the mechanism of lipid and lipoprotein binding of apoE3 NT domain, with the segment towards the C-terminal end likely triggering lipid interaction.

Improvement of stability and enzymatic activity by site-directed mutagenesis of E. coli asparaginase II

Bacterial asparaginases (EC 3.5.1.1) have attracted considerable attention because enzymes of this group are used in the therapy of certain forms of leukemia. Class II asparaginase from Escherichia coli (EcA), a homotetramer with a mass of 138kDa, is especially effective in cancer therapy. However, the therapeutic potential of EcA is impaired by the limited stability of the enzyme in vivo and by the induction of antibodies in the patients. In an attempt to modify the properties of EcA, several variants with amino acid replacements at subunit interfaces were constructed and characterized. Chemical and thermal denaturation analysis monitored by activity, fluorescence, circular dichroism, and differential scanning calorimetry showed that certain variants with exchanges that weaken dimer–dimer interactions exhibited complex denaturation profiles with active dimeric and/or inactive monomeric intermediates appearing at low denaturant concentrations. By contrast, other EcA variants showed considerably enhanced activity and stability as compared to the wild-type enzyme. Thus, even small changes at a subunit interface may markedly affect EcA stability without impairing its catalytic properties. Variants of this type may have a potential for use in the asparaginase therapy of leukemia.

Critical Evaluation of the Two-State Model Describing the Equilibrium Unfolding of the PI3K SH3 Domain by Time-Resolved Fluorescence Resonance Energy Transfer

It appears that equilibrium unfolding transitions of many small proteins can be described as two-state transitions, because the probes commonly used to measure such transitions cannot detect the underlying heterogeneity inherent in protein folding and unfolding reactions. Time-resolved fluorescence or Forster resonance energy transfer (TRFRET) measurements have the potential to uncover such heterogeneity and to test the cooperativity of protein folding reactions. Here, TRFRET measurements have been used to study the equilibrium unfolding of the SH3 domain of PI3 kinase. The single tryptophan residue (W53) was used as the FRET donor, and a covalently attached thionitrobenzoate moiety at either of two sites (C17 and C70) was used as the FRET acceptor. The individual lifetime and amplitude components estimated from fitting the fluorescence decay kinetics to the sum of three or four exponentials were determined over a range of denaturant concentrations. The equilibrium unfolding transitions reported by these components were found to be noncoincident, suggesting the presence of multiple conformations in equilibrium during the course of unfolding. Fluorescence lifetime distributions were also generated by the model-free maximum entropy method of analysis. Different segments of the protein were found to show differences in the expansion of the native state at low denaturant concentrations, suggestive of gradual structural transitions. The unfolded protein was found to swell at increasingly high denaturant concentrations. The evolution of the fluorescence lifetime distributions with increasing denaturant concentration was also found to be incompatible with a two-state equilibrium unfolding model.

Activation of human biliverdin-IXα reductase by urea: Generation of kinetically distinct forms during the unfolding pathway

Activation of enzymes by low concentrations of denaturants has been reported for a limited number of enzymes including lipocalin-type prostaglandin D synthase (L-PGDS) and adenylate kinase. During unfolding studies on human biliverdin-IXα reductase it was discovered that the enzyme is activated at low concentrations of urea. Under standard assay conditions the native enzyme displays pronounced substrate inhibition with biliverdin as variable substrate; however in the presence of 3M urea, the substrate inhibition is abolished and the enzyme exhibits Michaelian kinetics. When the initial rate kinetics with NADPH as variable substrate are conducted in 3M urea, the Vmax is increased 11-fold to 1.8μmol/min/mg and the apparent Km for biliverdin increases from 1 to 3μM. We report the existence of two kinetically distinct folded intermediates between the native and unfolded forms. When the period of incubation with urea was varied prior to measuring enzyme activity, the apparent Vmax was shown to decay to half that seen at zero time with a half life of 5.8minutes, while the apparent Km for NADPH remains constant at approximately 5μM. With NADH as cofactor the half life of the activated (A) form was 2.9minutes, and this form decays in 3M urea to a less active (LA) form. The apparent Km for NADH increases from 0.33mM to 2mM for the A and LA forms. These kinetically distinct species are reminiscent of the activity-enhanced and inactive forms of L-PGDS observed in the presence of urea and guanidine hydrochloride.

Direct Observation of Protein Unfolded State Compaction in the Presence of Macromolecular Crowding

Proteins fold and function in cellular environments that are crowded with other macromolecules. As a consequence of excluded volume effects, compact folded states of proteins should be indirectly stabilized due to destabilization of extended unfolded conformations. Here, we assess the role of excluded volume in terms of protein stability, structural dimensions and folding dynamics using a sugar-based crowding agent, dextran 20, and the small ribosomal protein S16 as a model system. To specifically address dimensions, we labeled the protein with BODIPY at two positions and measured Trp-BODIPY distances under different conditions. As expected, we found that dextran 20 (200 mg/ml) stabilized the variants against urea-induced unfolding. At conditions where the protein is unfolded, Förster resonance energy transfer measurements reveal that in the presence of dextran, the unfolded ensemble is more compact and there is residual structure left as probed by far-ultraviolet circular dichroism. In the presence of a crowding agent, folding rates are faster in the two-state regime, and at low denaturant concentrations, a kinetic intermediate is favored. Our study provides direct evidence for protein unfolded-state compaction in the presence of macromolecular crowding along with its energetic and kinetic consequences.

The Roles of Helix I and Strand 5A in the Folding, Function and Misfolding of α1-Antitrypsin

α1-Antitrypsin, the archetypal member of the serpin superfamily, is a metastable protein prone to polymerization when exposed to stressors such as elevated temperature, low denaturant concentrations or through the presence of deleterious mutations which, in a physiological context, are often associated with disease. Experimental evidence suggests that α1-Antitrypsin can polymerize via several alternative mechanisms in vitro. In these polymerization mechanisms different parts of the molecule are proposed to undergo conformational change. Both strand 5 and helix I are proposed to adopt different conformations when forming the various polymers, and possess a number of highly conserved residues however their role in the folding and misfolding of α1-Antitrypsin has never been examined. We have therefore created a range of α1Antitypsin variants in order to explore the role of these conserved residues in serpin folding, misfolding, stability and function. Our data suggest that key residues in helix I mediate efficient folding from the folding intermediate and residues in strand 5A ensure native state stability in order to prevent misfolding. Additionally, our data indicate that helix I is involved in the inhibitory process and that both structural elements undergo differing conformational rearrangements during unfolding and misfolding. These findings suggest that the ability of α1-Antitrypsin to adopt different types of polymers under different denaturing conditions may be due to subtle conformational differences in the transiently populated structures adopted prior to the I and M* states.

Stability of gp41 hairpin and helix bundle assembly probed by combined stacking and circular dichroic approaches

The envelope glycoprotein gp41 of HIV-1 undergoes structural rearrangement to form a helix hairpin during the virus-mediated fusion. Previous studies to investigate the folding and stability of hairpin did not monitor the end-to-end distance of the molecule. To directly probe the distance change, rhodamine dye was conjugated to the gp41 recombinant near the N- and C-terminal regions to detect the UV absorption and fluorescence intensity changes induced by the chemical denaturant guanidinium chloride (GdmCl). Using the singly- and doubly-labeled constructs allowed us to distinguish between the hairpin formation and protein oligomerization. A biphasic transition of helical structure for the wild type protein was revealed by circular dichroism measurements while unfolding of the hairpin occurred at 6M GdmCl. The relevance of our study to the fusion inhibitor for HIV-1 was borne out by results on the mutants at the positions within the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) regions. A monophasic transition at lower denaturant concentration was detected for the NHR mutant supporting the concept of differential stability of NHR and CHR helical structure. The conclusion that the observed unstacking of doubly-labeled variant arises principally from the intra-molecular dimers was drawn from the unstacking of the protein labeled in the loop. Remarkably, it is deduced that the hairpin is more stable than the CHR helical structure. A model for denaturation of the helix hairpin bundle was proposed from these results. The biological implications of the findings and further applications of the distance-based approach were discussed.

Nonnative Aggregation of an IgG1 Antibody in Acidic Conditions: Part 1. Unfolding, Colloidal Interactions, and Formation of High-Molecular-Weight Aggregates

Monomeric and aggregated states of an IgG1 antibody were characterized under acidic conditions as a function of solution pH (3.5-5.5). A combination of intrinsic/extrinsic fluorescence (FL), circular dichroism, calorimetry, chromatography, capillary electrophoresis, and laser light scattering were used to characterize unfolding, refolding, native colloidal interactions, aggregate structure and morphology, and aggregate dissociation. Lower pH led to larger net repulsive colloidal interactions, decreased thermal stability of Fc and Fab regions, and increased solubility of thermally accelerated aggregates. Unfolding of the Fab domains, and possibly the CH3 domain, was inferred as a key step in the formation of aggregation-prone monomers. High-molecular-weight soluble aggregates displayed nonnative secondary structure, had a semi-rigid chain morphology, and bound thioflavin T (ThT), consistent with at least a portion of the monomer forming amyloid-like structures. Soluble aggregates also formed during monomer refolding under conditions moving from high to low denaturant concentrations. Both thermally and chemically induced aggregates showed similar ThT binding and secondary structural changes, and were noncovalent based on dissociation in concentrated guanidine hydrochloride solutions. Changes in intrinsic FL during chemical versus thermal unfolding suggest a greater degree of structural change during chemical unfolding, although aggregation proceeded through partially unfolded monomers in both cases.

Folding studies of purified LamB protein, the maltoporin from the Escherichia coli outer membrane: Trimer dissociation can be separated from unfolding

The folding mechanisms for β-barrel membrane proteins present unique challenges because acquisition of both secondary and tertiary structure is coupled with insertion into the bilayer. For the porins in Escherichia coli outer membrane, the assembly pathway also includes association into homotrimers. We study the folding pathway for purified LamB protein in detergent and observe extreme hysteresis in unfolding and refolding, as indicated by the shift in intrinsic fluorescence. The strong hysteresis is not seen in unfolding and refolding a mutant LamB protein lacking the disulfide bond, as it unfolds at much lower denaturant concentrations than wild type LamB protein. The disulfide bond is proposed to stabilize the structure of LamB protein by clasping together the two sides of Loop 1 as it lines the inner cavity of the barrel. In addition we find that low pH promotes dissociation of the LamB trimer to folded monomers, which run at about one third the size of the native trimer during SDS PAGE and are much more resistant to trypsin than the unfolded protein. We postulate the loss at low pH of two salt bridges between Loop 2 of the neighboring subunit and the inner wall of the monomer barrel destabilizes the quaternary structure.