Characterization of monomeric substates of ascorbate oxidase

Ascorbate oxidase (AAO) is a large, multidomain, dimeric protein whose folding/unfolding pathway is characterized by a complex, multistep process. Here we used fluorescence correlation spectroscopy to demonstrate the formation of partially folded monomers by pH-induced full dissociation into subunits. Hence, the structural features of monomeric AAO could be studied by fluorescence and CD spectroscopy. We found that the monomers keep their secondary structure, whereas subtle conformational changes in the tertiary structure become apparent. AAO dissociation has also been studied when unfolding the protein by high hydrostatic pressure at different pH values. A strong protein concentration dependence was observed at pH 8, whereas the enzyme was either monomeric or dimeric at pH 10 and 6, respectively. The calculated volume change associated with the unfolding of monomeric AAO, ΔV ～ -55 mL·mol(-1), is in the range observed for most proteins of the same size. These findings demonstrate that partially folded monomeric species might populate the energy landscape of AAO and that the overall AAO stability is crucially controlled by a few quaternary interactions at the subunits' interface.     

Folding intermediates of the prion protein stabilized by hydrostatic pressure and low temperature

Prion diseases are associated with conformational conversion of the cellular prion protein, PrPC, into a misfolded form, PrPSc. We have investigated the equilibrium unfolding of the structured domain of recombinant murine prion protein, comprising residues 121-231 (mPrP-(121-231)). The equilibrium unfolding of mPrP-(121-231) by urea monitored by intrinsic fluorescence and circular dichroism (CD) spectroscopies indicated a two-state transition, without detectable folding intermediates. The fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5-disulfonic acid (bis-ANS) binds to native mPrP-(121-231), indicating exposure of hydrophobic domains on the protein surface. Increasing concentrations of urea (up to 4 M) caused the release of bound bis-ANS, whereas changes in intrinsic fluorescence and CD of mPrP took place only above 4 M urea. This indicates the existence of a partially unfolded conformation of mPrP, characterized by loss of bis-ANS binding and preservation of the overall structure of the protein, stabilized at low concentrations of urea. Hydrostatic pressure and low temperatures were also used to stabilize partially folded intermediates that are not detectable in the presence of chemical denaturants. Compression of mPrP to 3.5 kbar at 25 degrees C and pH 7 caused a slight decrease in intrinsic fluorescence emission and an 8-fold increase in bis-ANS fluorescence. Lowering the temperature to -9 degrees C under pressure reversed the decrease in intrinsic fluorescence and caused a marked (approximately 40-fold) increase in bis-ANS fluorescence. The increase in bis-ANS fluorescence at low temperatures was similar to that observed for mPrP at 1 atm at pH 4. These results suggest that pressure-assisted cold denaturation of mPrP stabilizes a partially folded intermediate that is qualitatively similar to the state obtained at acidic pH. Compression of mPrP in the presence of a subdenaturing concentration of urea stabilized another partially folded intermediate, and cold denaturation under these conditions led to complete unfolding of the protein. Possible implications of the existence of such partially folded intermediates in the folding of the prion protein and in the conversion to the PrPSc conformer are discussed.     

Folding of a de novo designed native-like four-helix bundle protein

The folding of a model native-like dimeric four-helix bundle protein, (alpha(2))(2), was investigated using guanidine hydrochloride, hydrostatic pressure, and low temperature. Unfolding by guanidine hydrochloride followed by circular dichroism and intrinsic fluorescence spectroscopy revealed a highly cooperative transition between the native-like and unfolded states, with free energy of unfolding determined from CD data, DeltaG(unf) = 14.3 +/- 0.8 kcal/mol. However, CD and intrinsic fluorescence data were not superimposable, indicating the presence of an intermediate state during the folding transition. To stabilize the folding intermediate, we used hydrostatic pressure and low temperature. In both cases, dissociation of the dimeric native-like (alpha(2))(2) into folded monomers (alpha(2)) was observed. van't Hoff analysis of the low temperature experiments, assuming a two-state dimer 171-monomer transition, yielded a free energy of dissociation of (alpha(2))(2) of DeltaG(diss) = 11.4 +/- 0.4 kcal/mol, in good agreement with the free energy determined from pressure dissociation experiments (DeltaG(diss) = 10.5 +/- 0.1 kcal/mol). Binding of the hydrophobic fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to the pressure- and cold-dissociated states of (alpha(2))(2) indicated the existence of molten-globule monomers. In conclusion, we demonstrate that the folding pathway of (alpha(2))(2) can be described by a three-state transition including a monomeric molten globule-like state.     

Metastable, partially folded states in the productive folding and in the misfolding and amyloid aggregation of proteins

Understanding the energetic and structural basis of protein folding in a physiological context may represent an important step toward the elucidation of protein misfolding and aggregation events that take place in several pathological states. In particular, investigation of the structure and thermodynamic properties of partially folded intermediate states involved in productive folding or in misfolding/aggregation may provide insight into these processes and suggest novel approaches to prevent misfolding in living organisms. This goal, however, has remained elusive, because such intermediates are often transient and correspond to metastable states that are little populated under physiological conditions. Characterization of these states requires their stabilization by means of manipulation of the experimental conditions, involving changes in temperature, pH, or addition of different types of denaturants. In the past few years, hydrostatic pressure has been increasingly used as a thermodynamic variable in the study of both protein folding and misfolding/aggregation transitions. Compared with other chemical or physical denaturing agents, a unique feature of pressure is its ability to induce subtle changes in protein conformation, allowing the stabilization of partially folded states that are usually not significantly populated under more drastic conditions. Much of the recent work in this field has focused on the characterization of folding intermediates, because they seem to be involved in a variety of disease-causing protein misfolding and aggregation reactions. Here, we review recent examples of the use of hydrostatic pressure as a tool to gain insight into the forces and energetics governing the productive folding or the misfolding and amyloid aggregation of proteions.     

Folding intermediates of a model three-helix bundle protein. Pressure and cold denaturation studies

The stability and equilibrium unfolding of a model three-helix bundle protein, alpha(3)-1, by guanidine hydrochloride (GdnHCl), hydrostatic pressure, and temperature have been investigated. The combined use of these denaturing agents allowed detection of two partially folded states of alpha(3)-1, as monitored by circular dichroism, intrinsic fluorescence emission, and fluorescence of the hydrophobic probe bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid). The overall free-energy change for complete unfolding of alpha(3)-1, determined from GdnHCl unfolding data, is +4.6 kcal/mol. The native state is stabilized by -1.4 kcal/mol relative to a partially folded pressure-denatured intermediate (I(1)). Cold denaturation at high pressure gives rise to a second partially (un)folded conformation (I(2)), suggesting a significant contribution of hydrophobic interactions to the stability of alpha(3)-1. The free energy of stabilization of the native-like state relative to I(2) is evaluated to be -2.5 kcal/mol. Bis-ANS binding to the pressure- and cold-denatured states indicates the existence of significant residual hydrophobic structure in the partially (un)folded states of alpha(3)-1. The demonstration of folding intermediates of alpha(3)-1 lends experimental support to a number of recent protein folding simulation studies of other three-helix bundle proteins that predicted the existence of such intermediates. The results are discussed in terms of the significance of de novo designed proteins for protein folding studies.     

Rough energy landscapes in protein folding: dimeric E. coli Trp repressor folds through three parallel channels

The folding mechanism of the dimeric Escherichia coli Trp repressor (TR) is a kinetically complex process that involves three distinguishable stages of development. Following the formation of a partially folded, monomeric ensemble of species, within 5 ms, folding to the native dimer is controlled by three kinetic phases. The rate-limiting step in each phase is either a non-proline isomerization reaction or a dimerization reaction, depending on the final denaturant concentration. Two approaches have been employed to test the previously proposed folding mechanism of TR through three parallel channels: (1) unfolding double-jump experiments demonstrate that all three folding channels lead directly to native dimer; and (2) the differential stabilization of the transition state for the final step in folding and the native dimer, by the addition of salt, shows that all three channels involve isomerization of a dimeric species. A refined model for the folding of Trp repressor is presented, in which all three channels involve a rapid dimerization reaction between partially folded monomers followed by the isomerization of the dimeric intermediates to yield native dimer. The ensemble of partially folded monomers can be captured at equilibrium by low pH; one-dimensional proton NMR spectra at pH 2.5 demonstrate that monomers exist in two distinct, slowly interconverting conformations. These data provide a potential structural explanation for the three-channel folding mechanism of TR: random association of two different monomeric forms, which are distinguished by alternative packing modes of the core dimerization domain and the DNA-binding, helix-turn-helix, domain. One, perhaps both, of these packing modes contains non-native contacts.     

Cold rescue of the thermolabile tailspike intermediate at the junction between productive folding and off-pathway aggregation.

Off-pathway intermolecular interactions between partially folded polypeptide chains often compete with correct intramolecular interactions, resulting in self-association of folding intermediates into the inclusion body state. Intermediates for both productive folding and off-pathway aggregation of the parallel beta-coil tailspike trimer of phage P22 have been identified in vivo and in vitro using native gel electrophoresis in the cold. Aggregation of folding intermediates was suppressed when refolding was initiated and allowed to proceed for a short period at 0 degrees C prior to warming to 20 degrees C. Yields of refolded tailspike trimers exceeding 80% were obtained using this temperature-shift procedure, first described by Xie and Wetlaufer (1996, Protein Sci 5:517-523). We interpret this as due to stabilization of the thermolabile monomeric intermediate at the junction between productive folding and off-pathway aggregation. Partially folded monomers, a newly identified dimer, and the protrimer folding intermediates were populated in the cold. These species were electrophoretically distinguished from the multimeric intermediates populated on the aggregation pathway. The productive protrimer intermediate is disulfide bonded (Robinson AS, King J, 1997, Nat Struct Biol 4:450-455), while the multimeric aggregation intermediates are not disulfide bonded. The partially folded dimer appears to be a precursor to the disulfide-bonded protrimer. The results support a model in which the junctional partially folded monomeric intermediate acquires resistance to aggregation in the cold by folding further to a conformation that is activated for correct recognition and subunit assembly.     

Thermodynamic characterization of an intermediate state of human growth hormone.

The thermal denaturation of recombinant human growth hormone (rhGH) was studied by differential scanning calorimetry and circular dichroism spectroscopy (CD). The thermal unfolding is reversible only below pH 3.5, and under these conditions a single two-state transition was observed between 0 and 100 degrees C. The magnitudes of the deltaH and deltaCp of this transition indicate that it corresponds to a partial unfolding of rhGH. This is also supported by CD data, which show that significant secondary structure remains after the unfolding. Above pH 3.5 the thermal denaturation is irreversible due to the aggregation of rhGH upon unfolding. This aggregation is prevented in aqueous solutions of alcohols such as n-propanol, 2-propanol, or 1,2-propanediol (propylene glycol), which suggests that the self-association of rhGH is caused by hydrophobic interactions. In addition, it was found that the native state of rhGH is stable in relatively high concentrations of propylene glycol (up to 45% v/v at pH 7-8 or 30% at pH 3) and that under these conditions the thermal unfolding is cooperative and corresponds to a transition from the native state to a partially folded state, as observed at acidic pH in the absence of alcohols. In higher concentrations of propylene glycol, the tertiary structure of rhGH is disrupted and the cooperativity of the unfolding decreases. Moreover, the CD and DSC data indicate that a partially folded intermediate with essentially native secondary structure and disordered tertiary structure becomes significantly populated in 70-80% propylene glycol.     

Alpha-synuclein multistate folding thermodynamics: implications for protein misfolding and aggregation

Alpha-synuclein aggregation has been tightly linked with the pathogenesis of Parkinson's disease and other neurodegenerative disorders. Despite the protein's putative function in presynaptic vesicle regulation, the roles of lipid binding in modulating alpha-synuclein conformations and the aggregation process remain to be fully understood. This study focuses on a detailed thermodynamic characterization of monomeric alpha-synuclein folding in the presence of SDS, a well-studied lipid mimetic. Far-UV CD spectroscopy was employed for detection of conformational transitions induced by SDS, temperature, and pH. The data we present here clearly demonstrate the multistate nature of alpha-synuclein folding, which involves two predominantly alpha-helical partially folded thermodynamic intermediates that we designate as F (most folded) and I (intermediately folded) states. Likely structures of these alpha-synuclein conformational states are also discussed. These partially folded forms can exist in the presence of either monomeric or micellar forms of SDS, which suggests that alpha-synuclein has an intrinsic propensity for adopting multiple alpha-helical structures even in the absence of micelle or membrane binding, a feature that may have implications for its biological activity and toxicity. Additionally, we discuss the relation between alpha-synuclein three-state folding and its aggregation, within the context of isothermal titration calorimetry and transmission electron microscopy measurements of SDS-initiated oligomer formation.     

Predissociated dimers and molten globule monomers in the equilibrium unfolding of yeast glutathione reductase

The equilibrium unfolding of dimeric yeast glutathione reductase (GR) by guanidine hydrochloride (GdnHCl) was investigated. Unfolding was monitored by a variety of techniques, including intrinsic fluorescence emission, anisotropy and iodide quenching measurements, far-ultraviolet circular dichroism and thiol reactivity measurements. At 1 M GdnHCl, one thiol group of GR became accessible to modification with 5,5′-dithiobis-(2-nitrobenzoic) acid (DTNB), whereas no changes could be detected in the spectroscopic properties (fluorescence, circular dichroism) of the protein. Between 2 and 3 M GdnHCl, two partially folded intermediate states possessing flexible tertiary structures (revealed by fluorescence data) but compact secondary structures (as indicated by circular dichroism measurements) were identified. The quaternary structure of GR in the presence of GdnHCl was also investigated by size-exclusion liquid chromatography. These results indicated the presence of an expanded predissociated dimer at 2.5 M GdnHCl and partially folded monomers at 3 M GdnHCl. Taken together, these results suggest the existence of two molten-globule-like intermediate species (one dimeric and one monomeric) in the unfolding of GR. The results are discussed in terms of the mechanism of GR folding and dimerization.     

Reversible denaturation of the gene V protein of bacteriophage f1

The guanidine hydrochloride (GuHCl)-induced denaturation of the gene V protein of bacteriophage f1 has been studied, using the chemical reactivity of a cysteine residue that is buried in the folded protein and the circular dichroism (CD) at 211 and 229 nm as measures of the fraction of polypeptide chains in the folded form. It is found that this dimeric protein unfolds in a single cooperative transition from a folded dimer to two unfolded monomers. A folded, monomeric form of the gene V protein was not detected at equilibrium. The kinetics of unfolding of the gene V protein in 3 M GuHCl and the refolding in 2 M GuHCl are also consistent with a transition between a folded dimer and two unfolded monomers. The GuHCl concentration dependence of the rates of folding and unfolding suggests that the transition state for folding is near the folded conformation.     

Dynamic properties of pH-dependent structural organization of the amyloidogenic β-protein (1-40)

The structural organization of the amyloidogenic β-proteins containing 40 amino acid residues (Aβ40) was studied by the high temperature molecular dynamics simulations in the acidic (pH~3) and basic (pH~8) pH regions. The obtained data suggest that the central Ala21-Gly29 segment of Aβ40, can adopt folded and partially unfolded structures. At the basic pH, this segment forms folded structures, stabilized by electrostatic interactions and hydrogen bonds. At the acidic pH, it forms partially unfolded structures. Two other segments flanking to the central segment exhibit the propensity to adopt unstable inter-converting α-helical, 310-helical and turn-like structures. One of these segments is comprised of the Ala30-Val36 residues at both of the considered pHs. The second segment is comprised of the Glu11-Phe20 at the basic pH and of the Glu11-Val24 residues at the acidic pHs. The revealed pH-dependent structuration of the Aβ40 allowed us to suggest a possible scenario for initial Aβ aggregation. According to this scenario, the occurrence of the partially unfolded states of the Ala21-Gly29 segment plays main role in the Aβ oligomerization process.     

Aromatic residues engineered into the beta-turn nucleation site of ubiquitin lead to a complex folding landscape, non-native side-chain interactions, and kinetic traps

The fast folding of small proteins is likely to be the product of evolutionary pressures that balance the search for native-like contacts in the transition state with the minimum number of stable non-native interactions that could lead to partially folded states prone to aggregation and amyloid formation. We have investigated the effects of non-native interactions on the folding landscape of yeast ubiquitin by introducing aromatic substitutions into the beta-turn region of the N-terminal beta-hairpin, using both the native G-bulged type I turn sequence (TXTGK) as well as an engineered 2:2 XNGK type I' turn sequence. The N-terminal beta-hairpin is a recognized folding nucleation site in ubiquitin. The folding kinetics for wt-Ub (TLTGK) and the type I' turn mutant (TNGK) reveal only a weakly populated intermediate, however, substitution with X = Phe or Trp in either context results in a high propensity to form a stable compact intermediate where the initial U-->I collapse is visible as a distinct kinetic phase. The introduction of Trp into either of the two host turn sequences results in either complex multiphase kinetics with the possibility of parallel folding pathways, or formation of a highly compact I-state stabilized by non-native interactions that must unfold before refolding. Sequence substitutions with aromatic residues within a localized beta-turn capable of forming non-native hydrophobic contacts in both the native state and partially folded states has the undesirable consequence that folding is frustrated by the formation of stable compact intermediates that evolutionary pressures at the sequence level may have largely eliminated.     

Acid-Induced Formation of Molten Globule States in the Wild Type <Emphasis Type="Italic">Escherichia coli</Emphasis> 5-Enolpyruvylshikimate 3-Phosphate Synthase and its Three Mutated Forms: G96A,A183T and G96A/A183T

Recent advances in protein chemistry have led to progress in the understanding of protein folding and properties of possible intermediates during the folding of proteins. The molten globule (MG) state, a major intermediate of protein folding, has a denatured state with native-like secondary structure. In the present work, the acid-induced unfolding of wild type Escherichia coli 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) and its three different variants (G96A, A183T and G96A/A183T) were studied by far- and near-UV circular dichroism (CD), intrinsic fluorescent emission spectroscopy and 1-anilino naphthalene-8-sulfonate (ANS) binding. At pH < 3.0, these EPSPS variants acquire partially folded state, which show the characteristics of the MG state, e.g., a drastic reduction of defined tertiary structure and almost no change in the secondary structure. ANS binding experiments show that hydrophobic surface of these variants is exposed to a greater extent in comparison to the native form, at acidic pH. Wild type, G96A, A183T and G96A/A183T acquire MG states at pH 2.0, 1.5, 3.0 and 3.0, respectively, which show that pH stability of MG state of G96A has increased in comparison to wild type; and pH stability of MG states of two other mutants is lower than that of the wild type. The results suggest that there is a direct relationship between stability of protein and pH stability of its folding intermediates.     

Dynamic properties of pH-dependent structural organization of the amyloidogenic ��-protein (1�C40)

The structural organization of the amyloidogenic β-protein containing 40 amino acid residues (Aβ40) was studied by the high temperature molecular dynamics simulations in the acidic (pH ∼ 3) and basic (pH ∼ 8) pH regions. The obtained data suggest that the central Ala21-Gly29 segment of Aβ40 can adopt folded and partially unfolded structures. At the basic pH, this segment forms folded structures stabilized by electrostatic interactions and hydrogen bonds. At the acidic pH, it forms partially unfolded structures. Two other segments flanking to the central segment exhibit the propensity to adopt unstable interconverting α-helical, 3₁₀-helical and turn-like structures. One of these segments is comprised of the Ala30-Val36 residues at both of the considered pHs. The second segment is comprised of the Glu11-Phe20 at the basic pH and of the Glu11-Val24 residues at the acidic pHs. The revealed pH-dependent structuration of the Aβ40 allowed us to suggest a possible scenario for initial Aβ aggregation. According to this scenario, the occurrence of the partially unfolded states of the Ala21-Gly29 segment plays main role in the Aβ oligomerization process.Key words: amyloid-β protein, Alzheimer disease, oligomerization, fibril, electrostatic interactions, molecular dynamics simulations     

Alternatively folded states of an immunoglobulin

Well-defined, non-native protein structures of low stability have been increasingly observed as intermediates in protein folding or as equilibrium structures populated under specific solvent conditions. These intermediate structures, frequently referred to as molten globule states, are characterized by the presence of secondary structure, a lack of significant tertiary contacts, increased hydrophobicity and partial specific volume as compared to native structures, and low cooperativity in thermal unfolding. The present study demonstrates that under acidic conditions (pH less than 3) the antibody MAK33 can assume a folded stable conformation. This A-state is characterized by a high degree of secondary structure, increased hydrophobicity, a native-like maximum wavelength of fluorescence emission, and a tendency toward slow aggregation. A prominent feature of this low-pH conformation is the stability against denaturant and thermal unfolding that is manifested in highly cooperative reversible phase transitions indicative of the existence of well-defined tertiary contacts. These thermodynamic results are corroborated by the kinetics of folding from the completely unfolded chain to the alternatively folded state at pH 2. The given data suggest that MAK33 at pH 2 adopts a cooperative structure that differs from the native immunoglobulin fold at pH 7. This alternatively folded state exhibits certain characteristics of the molten globule but differs distinctly from it by its extraordinary structural stability that is characteristic for native protein structures.     

Hydrophobic core variant ubiquitin forms a molten globule conformation at acidic pH

The conformational properties of hydrophobic core variant ubiquitin (Val26 to Ala mutation) in an acidic solution were studied. The intrinsic tryptophan fluorescence emission spectrum, far-UV and near-UV circular dichroic spectra, the fluorescence emission spectrum of 8-anilinonaphthalene-1-sulfonic acid in the presence of V26A ubiquitin, and urea-induced unfolding measurements indicate this variant ubiquitin to be in the partially folded molten globule conformation in solution at pH 2. The folding kinetics from molten globule to the native state was nearly identical to those from the unfolded state to the native state. This observation suggests that the equilibrium molten globule state of hydrophobic core variant ubiquitin is an on-pathway folding intermediate.     

Protein folding and aggregation: two sides of the same coin in the condensation of proteins revealed by pressure studies

Hydrostatic pressure can be considered as "thermodynamic tweezers" to approach the protein folding problem and to study the cases when folding goes wrong leading to the protein folding disorders. The main outcome of the use of high pressure in this field is the stabilization of folding intermediates such as partially folded conformations, thus allowing us to characterize their structural properties. Because partially folded intermediates are usually at the intersection between productive and off-pathway folding, they may give rise to misfolded proteins, aggregates and amyloids that are involved in many neurodegenerative diseases, such as transmissible spongiform encephalopathies, Alzheimer's disease, Parkinson's disease and Huntington's disease. Of particular interest is the use of hydrostatic pressure to unveil the structural transitions in prion conversion and to populate possible intermediates in the folding/unfolding pathway of the prion protein. The main hypothesis for prion diseases proposes that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform, the PrP(Sc) (from scrapie). It has been demonstrated that hydrostatic pressure affects the balance between the different prion species. The last findings on the application of high pressure on amyloidogenic proteins will be discussed here as regards to their energetic and volumetric properties. The use of high pressure promises to contribute to the identification of the underlying mechanisms of these neurodegenerative diseases and to develop new therapeutic approaches.     

Structure and function in bacteriorhodopsin: the effect of the interhelical loops on the protein folding kinetics

The loops connecting the seven transmembrane helices of bacteriorhodopsin have each been replaced in turn by structureless linkers of Gly-Gly-Ser repeat sequences, and the effect on the protein folding kinetics has been determined. An SDS-denatured state of each loop mutant bacterio-opsin was folded in l-alpha-1,2-dihexanoylphosphatidylcholine/l-alpha-1,2-dimyristoylphosphatidylcholine micelles, containing retinal, to give functional bacteriorhodopsin. Stopped-flow mixing was used to initiate the folding reaction, giving a time resolution of milliseconds, and changes in protein fluorescence were used to monitor folding. All loop mutant proteins folded according to the same reaction scheme as wild-type protein. The folding kinetics of the AB, BC and DE loop mutants were the same as wild-type protein, despite the blue-shifted chromophore band of the BC loop mutant bR state. A partially folded apoprotein intermediate state of the AB loop mutant did however appear to decay in the absence of retinal. The most significant effects on the folding kinetics were seen for mutant protein with structureless linkers in place of the CD, EF and FG loops. The rate-limiting apoprotein folding step of the CD loop mutant was about ten times slower than wild-type, whilst that of the EF loop mutant was almost four times slower than wild-type. Wild-type behaviour was observed for the other folding and retinal binding events of the CD and EF loop mutant proteins. These effects of the CD and EF loop mutations on apoprotein folding correlate with the fact that these two loop mutants also have the least stable, partially folded apoprotein intermediate of all the loop mutants, and are the most affected by a decrease in lipid lateral pressure. In contrast, the FG loop mutant exhibited wild-type apoprotein folding, but altered covalent binding of retinal and final folding to bacteriorhodopsin. This correlates with the fact that the FG loop mutant bacteriorhodopsin is the most susceptible to denaturation by SDS of all the loop mutants, but its partially folded apoprotein intermediate is more stable than that of the CD and EF mutants. Thus the CD and EF loops may contribute to the transition state for the rate-limiting apoprotein folding step and the FG loop to that for final folding and covalent binding of retinal.     

Conformation of P22 tailspike folding and aggregation intermediates probed by monoclonal antibodies.

The partitioning of partially folded polypeptide chains between correctly folded native states and off-pathway inclusion bodies is a critical reaction in biotechnology. Multimeric partially folded intermediates, representing early stages of the aggregation pathway for the P22 tailspike protein, have been trapped in the cold and isolated by nondenaturing polyacrylamide gel electrophoresis (PAGE) (speed MA, Wang DIC, King J. 1995. Protein Sci 4:900-908). Monoclonal antibodies against tailspike chains discriminate between folding intermediates and native states (Friguet B, Djavadi-Ohaniance L, King J, Goldberg ME. 1994. J Biol Chem 269:15945-15949). Here we describe a nondenaturing Western blot procedure to probe the conformation of productive folding intermediates and off-pathway aggregation intermediates. The aggregation intermediates displayed epitopes in common with productive folding intermediates but were not recognized by antibodies against native epitopes. The nonnative epitope on the folding and aggregation intermediates was located on the partially folded N-terminus, indicating that the N-terminus remained accessible and nonnative in the aggregated state. Antibodies against native epitopes blocked folding, but the monoclonal directed against the N-terminal epitope did not, indicating that the conformation of the N-terminus is not a key determinant of the productive folding and chain association pathway.