蛋白质的氧化重折叠

经过近几十年来广泛而深入的研究,蛋白质氧化重折叠的机制已得到相当详细的阐明。1在已研究过的蛋白质中,大多数蛋白质都是沿着多途径而非单一、特定的途径进行氧化重折叠,这与折叠能量景观学说是一致的。2正是氨基酸残基间的天然相互作用而不是非天然的相互作用控制蛋白质的折叠过程。这一结论与含非天然二硫键的折叠中间体在牛胰蛋白酶抑制剂（BPTI）折叠中所起的重要作用并非相互排斥,因为后者仅仅是进行链内二硫键重排的化学反应所必需,与控制肽链折叠无直接关系。3根据对BPTI的研究,二硫键曾被认为仅仅具有稳定蛋白质天然结构的作用,既不决定折叠途径也不决定其三维构象。这一观点不适用于其它蛋白质。对凝乳酶原的研究表明,天然二硫键的形成是恢复天然构象的前提。天然二硫键的形成与肽键的正确折叠相辅相成,更具有普遍意义。4在氧化重折叠的早期,二硫键的形成基本上是一个随机过程,随着肽链的折叠二硫键的形成越来越受折叠中间体构象的限制。提高重组蛋白质的复性产率是生物技术领域中的一个巨大的挑战。除了分子聚集外,在折叠过程中所形成的二硫键错配分子是导致低复性率的另一个主要原因。氧化重折叠机制的阐明为解决此问题提供了有益的启示。如上所述,在折叠的后期,二硫键的形成决定于折叠中间体的构象,类天然、有柔性的结构有利于天然二硫键形成和正确折叠,具有这类结构的分子为有效的折叠中间体,最终都能转变为天然产物;而无效折叠中间体往往具有稳定的结构,使巯基、二硫键内埋妨碍二硫键重排,并因能垒的障碍不利于进一步折叠。因此,降低无效折叠中间体的稳定性使之转变为有效折叠中间体是提高含二硫键蛋白质复性率的一条基本原则,实验证明,碱性pH、低温、降低蛋白质稳定性的试剂、蛋白质二硫键异构酶、改变蛋白质一级结构是实现这一原则的有效手段。此外,这里还就氧化重折叠的基础和应用研究的前景进行了讨论。     

Molecular basis of co-operativity in protein folding. III. Structural identification of cooperative folding units and folding intermediates.

The hierarchical partition function formalism for protein folding developed earlier has been extended through the use of three-dimensional polar and apolar contact plots. For each amino acid residue in the protein, these plots indicate the apolar and polar surfaces that are buried from the solvent, the identity of all amino acid residues that contribute to this shielding, and the magnitude of their contributions. These contact plots are then used to examine the distribution of the free energy of stabilization throughout the protein molecule. Analysis of these data allows identification of co-operative folding units and their hierarchical levels, and the identification of partially folded intermediates with a significant probability of being populated. The overall folding/unfolding thermodynamics of 12 globular proteins, for which crystallographic and experimental thermodynamics are available, is predicted within error. An energetic classification of partially folded intermediates is presented and the results compared to those cases for which structural and thermodynamic experimental information is available. Four different types of partially folded states and their structural energies are considered. (1) Local intermediates, in which only a local region of the protein loses secondary and tertiary interactions, while the rest of the protein remains intact. (2) Global intermediates, corresponding to the standard molten globule definition, in which significant secondary structure is maintained but native-like tertiary structure contacts are disrupted. (3) Extended intermediates characterized by the existence of secondary structure elements (e.g. alpha-helices) exposed to solvent. (4) Folding intermediates in proteins with two structural domains. The structure and energetics of folding intermediates of apo-myoglobin, alpha-lactalbumin, phosphoglycerate kinase and arabinose-binding protein are considered in detail.     

Association of partially-folded intermediates of staphylococcal nuclease induces structure and stability

Staphylococcal nuclease forms three different partially-folded intermediates at low pH in the presence of low to moderate concentration of anions, differing in the amount of secondary structure, globularity, stability, and compactness. Although these intermediates are monomeric at low protein concentration (< or =0.25 mg/mL), increasing concentrations of protein result in the formation of dimers and soluble oligomers, ultimately leading to larger insoluble aggregates. Unexpectedly, increasing protein concentration not only led to association, but also to increased structure of the intermediates. The secondary structure, stability, and globularity of the two less-ordered partially-folded intermediates (A1 and A2) were substantially increased upon association, suggesting that aggregation induces structure. An excellent correlation was found between degree of association and amount of structure measured by different techniques, including circular dichroism, fluorescence, Fourier transform infrared spectroscopy (FTIR), and small-angle X-ray scattering. The associated states were also substantially more stable toward urea denaturation than the monomeric forms. A mechanism is proposed, in which the observed association of monomeric intermediates involves intermolecular interactions which correspond to those found intramolecularly in normal folding to the native state.     

Native and non-native intermediates in the BPTI folding pathway.

Recent studies of the disulfide-bonded intermediates in the refolding of bovine pancreatic trypsin inhibitor (BPTI) indicate that the most stable intermediates can take on much or all of the structure of the fully folded protein. Native-like structure in the intermediates probably causes steric inhibition of direct sequential formation of the three disulfides found in the native protein, thus accounting for the role of intramolecular rearrangements in the folding mechanism of this small protein.     

Hydrogen exchange methods to study protein folding

The measurement of amino acid-resolved hydrogen exchange (HX) has provided the most detailed information so far available on the structure and properties of protein folding intermediates. Direct HX measurements can define the structure of tenuous molten globule forms that are generally inaccessible to the usual crystallographic and NMR methods (C. Redfield review in this issue). HX pulse labeling methods can specify the structure, stability and kinetics of folding intermediates that exist for less than 1 s during kinetic folding. Native state HX methods can detect and characterize folding intermediates that exist as infinitesimally populated high energy excited state forms under native conditions. The results obtained in these ways suggest principles that appear to explain the properties of partially folded intermediates and how they are organized into folding pathways. The application of these methods is detailed here.     

The influence of X-rays on the structural studies of peroxide-derived myoglobin intermediates

In recent years, the awareness of potential radiation damage of metal centers in protein crystals during crystallographic data collection has received increasing attention. The radiation damage can lead to radiation-induced changes and reduction of the metal sites. One of the research fields where these concerns have been comprehensively addressed is the study of the reaction intermediates of the heme peroxidase and oxygenase reaction cycles. For both the resting states and the high-valent intermediates, the X-rays used in the structure determination have given undesired side effects through radiation-induced changes to the trapped intermediates. However, X-rays have been used to generate and trap the peroxy/hydroperoxy state in crystals. In this review, the structural work and the influence of X-rays on these intermediates in myoglobin are summarized and viewed in light of analogous studies on similar intermediates in peroxidases and oxygenases.     

Probing the folding pathways of long R(3) insulin-like growth factor-I (LR(3)IGF-I) and IGF-I via capture and identification of disulfide intermediates by cyanylation methodology and mass spectrometry

This report describes an integrated investigation of the refolding and reductive unfolding of insulin-like growth factor (IGF-I) and its variant, long R(3) IGF-I (LR(3)IGF-I), which has a Glu(3) to Arg(3) substitution and a hydrophobic 13-amino acid N-terminal extension. The refolding performed in glutathione redox buffer was quenched at different time points by adjusting the pH to 2.0-3.0 with a 1 N HCl solution of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, which trapped intermediates via cyanylation of free sulfhydryl groups. The disulfide structure of the intermediates was determined by chemical cleavage followed by mass mapping with mass spectrometry. Six refolding intermediates of IGF-I and three refolding intermediates of LR(3)IGF-I were isolated and characterized. Folding pathways of IGF-I and LR(3)IGF-I are proposed based on the time-dependent distribution and disulfide structure of the corresponding trapped intermediates. Similarities and differences in the refolding behavior of IGF-I and LR(3)IGF-I are discussed.     

Conformational forces affecting the folding pathways of dendrotoxins I and K from black mamba venom

The conformations of the major intermediates trapped during the folding of dendrotoxins I and K from venom of black mamba snakes, have been investigated by circular-dichroism spectroscopy. Local alterations to the native, folded conformations are observed in toxins I and K and in a protein of similar sequence, bovine pancreatic trypsin inhibitor. The inability of intermediates (30-51, 14-38) to complete refolding by forming directly the 5-55 disulphide bond is explained. The following observations on the role of secondary structure in the folding of the three proteins are of interest. 1. It is not necessary for the three proteins to acquire elements of secondary structure at the same stage of folding in order to attain similar three-dimensional conformations. 2. The stability of the final folded state is not directly correlated to an early appearance of secondary structure. 3. The degree of secondary structure already present in intermediates (30-51) seems to determine the pathway of refolding preferred by the corresponding protein.     

An evidence for the equilibrium unfolding intermediates of ribonuclease A by tritium labeling method

A formation of a molten globule in the unfolding of ribonuclease A could be considered as an evidence supporting a hypothesis on the existence of such intermediates on the pathway of a protein folding. Using a novel technique (tritium labeling method) we have showed that the ribonuclease A equilibrium unfolding in urea and guanidinium chloride (GuCl) solutions proceeds through a formation of intermediates whose properties (compactness, retention of the larger part hydrophobic core, secondary structure, and native-like folding pattern) correspond to the fundamental characteristics of the molten globule state. The both intermediates are the “wet” molten globules (the globule interior contains the water molecules). The results reveal the noticeable distinctions in intermediates structure, first of all, in the extent of their compactness. The urea intermediate is less compact than that in GuCl. It is shown that the refolding of the protein denatured by GuCl results in the formation of the intermediate which enzyme activity is virtually the same as the activity of the native protein.     

Detection of intermediate species in the refolding of bovine trypsinogen

The mixed disulfide of bovine trypsinogen and glutathione was refolded at pH 8.6 and 4 degrees C with a mixture of 3 mM cysteine and 1 mM cystine catalyzing disulfide interchange. The folding process was monitored by analysis of quenched samples with isoelectric focusing and size-exclusion chromatography. Isoelectric focusing showed a progressive change from a pI of 5.2 for the mixed disulfide derivative to a pI of 9.3 for native trypsinogen. A number of principal intermediates were detected as a function of the refolding time. These intermediates were also separated and further characterized by size-exclusion chromatography on columns of TSK G2000 SW operated in the high-performance liquid chromatographic mode. Rechromatography of a series of sequential fractions taken from the parental peak was necessary to resolve and characterize the principal intermediates. The loss of glutathione moieties produced a partly folded structure with an apparent hydrodynamic volume (Stokes radius, Rs) of 33.9 A. These structures became compact with time, and more intermediates were detected between 33.9 and 29.2 A. Finally, a change in conformation, resembling a two-state transition, changed the molecules of Rs 29.2 to the compact structure of native trypsinogen (22.4 A). The rate of formation of the native structure was determined from the progress curves derived from isoelectric focusing and size-exclusion chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transition of a Compact Intermediate State of Pea Lectin Under the Influence of Different Molecular Weight Polyethylene Glycols

The compact intermediate of the pea lectin found to exist at pH 2.4 was treated with low (PEG-400), medium (PEG-4000) and high (PEG-20,000) molecular weight PEGs. The changes occurring in the secondary structure of the protein were monitored by CD spectropolarimetry in the far-UV range, intrinsic fluorescence was used as a probe to observe the changes in the tertiary structure which is reflected by the changes in the tryptophan environment, further ANS binding studies were made to know the extent of exposure of the hydrophobic patches which is again indicative of the overall changes occurring in the tertiary structure of the protein. It was found that the three PEGs altered the secondary as well as tertiary structure of the pH 2.4 intermediate leading to the formation of three different intermediates. The intermediates were found to have non-native secondary structure as well as non-native tertiary structure. The intermediate formed by the action of PEG-400 was due to the induction of secondary and tertiary structure while the intermediates formed under the influence of PEG-4000 and PEG-20,000 were due to loss in secondary structure and rearrangement in tertiary structure. Also the ANS binding studies showed the absence of any MG or MG-like structures formed in the folding /unfolding pathway induced by PEGs.     

Observing a late folding intermediate of Ubiquitin at atomic resolution by NMR

The study of intermediates in the protein folding pathway provides a wealth of information about the energy landscape. The intermediates also frequently initiate pathogenic fibril formations. While observing the intermediates is difficult due to their transient nature, extreme conditions can partially unfold the proteins and provide a glimpse of the intermediate states. Here, we observe the high resolution structure of a hydrophobic core mutant of Ubiquitin at an extreme acidic pH by nuclear magnetic resonance (NMR) spectroscopy. In the structure, the native secondary and tertiary structure is conserved for a major part of the protein. However, a long loop between the beta strands β3 and β5 is partially unfolded. The altered structure is supported by fluorescence data and the difference in free energies between the native state and the intermediate is reflected in the denaturant induced melting curves. The unfolded region includes amino acids that are critical for interaction with cofactors as well as for assembly of poly‐Ubiquitin chains. The structure at acidic pH resembles a late folding intermediate of Ubiquitin and indicates that upon stabilization of the protein's core, the long loop converges on the core in the final step of the folding process.     

Multiple unfolding intermediates of human placental alkaline phosphatase in equilibrium urea denaturation.

Alkaline phosphatase is an enzyme with a typical alpha/beta hydrolase fold. The conformational stability of the human placental alkaline phosphatase was examined with the chemical denaturant urea. The red shifts of fluorescence spectra show a complex unfolding process involving multiple equilibrium intermediates indicating differential stability of the subdomains of the enzyme. None of these unfolding intermediates were observed in the presence of 83 mM NaCl, indicating the importance of ionic interactions in the stabilization of the unfolding intermediates. Guanidinium chloride, on the other hand, could stabilize one of the unfolding intermediates, which is not a salt effect. Some of the unfolding intermediates were also observed in circular dichroism spectroscopy, which clearly indicates steady loss of helical structure during unfolding, but very little change was observed for the beta strand content until the late stage of the unfolding process. The enzyme does not lose its phosphate-binding ability after substantial tertiary structure changes, suggesting that the substrate-binding region is more resistant to chemical denaturant than the other structural domains. Global analysis of the fluorescence spectral change demonstrated the following folding-unfolding process of the enzyme: N <--> I(1) <--> I(2) <--> I(3) <--> I(4) <--> I(5) <--> D. These discrete intermediates are stable at urea concentrations of 2.6, 4.1, 4.7, 5.5, 6.6, and 7.7 M, respectively. These intermediates are further characterized by acrylamide and/or potassium iodide quenching of the intrinsic fluorescence of the enzyme and by the hydrophobic probes, 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid. The stepwise unfolding process was interpreted by the folding energy landscape in terms of the unique structure of the enzyme. The rigid central beta-strand domain is surrounded by the peripheral alpha-helical and coil structures, which are marginally stable toward a chemical denaturant.     

All or none fibrillogenesis of a prion peptide.

Amyloid proteins and peptides comprise a diverse group of molecules that vary both in size and amino-acid sequence, yet assemble into amyloid fibrils that have a common core structure. Kinetic studies of amyloid fibrillogenesis have revealed that certain amyloid proteins form oligomeric intermediates prior to fibril formation. We have investigated fibril formation with a peptide corresponding to residues 195-213 of the human prion protein. Through a combination of kinetic and equilibrium studies, we have found that the fibrillogenesis of this peptide proceeds as an all-or-none reaction where oligomeric intermediates are not stably populated. This variation in whether oligomeric intermediates are stably populated during fibril formation indicates that amyloid proteins assemble into a common fibrillar structure; however, they do so through different pathways.     

Structure of stable protein folding intermediates by equilibrium phi-analysis: the apoflavodoxin thermal intermediate

Protein intermediates in equilibrium with native states may play important roles in protein dynamics but, in cases, can initiate harmful aggregation events. Investigating equilibrium protein intermediates is thus important for understanding protein behaviour (useful or pernicious) but it is hampered by difficulties in gathering structural information. We show here that the phi-analysis techniques developed to investigate transition states of protein folding can be extended to determine low-resolution three-dimensional structures of protein equilibrium intermediates. The analysis proposed is based solely on equilibrium data and is illustrated by determination of the structure of the apoflavodoxin thermal unfolding intermediate. In this conformation, a large part of the protein remains close to natively folded, but a 40 residue region is clearly unfolded. This structure is fully consistent with the NMR data gathered on an apoflavodoxin mutant designed specifically to stabilise the intermediate. The structure shows that the folded region of the intermediate is much larger than the proton slow-exchange core at 25 degrees C. It also reveals that the unfolded region is made of elements whose packing surface is more polar than average. In addition, it constitutes a useful guide to rationally stabilise the native state relative to the intermediate state, a far from trivial task.     

Capturing time-resolved changes in molecular structure by negative staining

Imaging structural intermediates of biological processes is a key step in understanding biological function. Because intermediates are commonly short-lived, lasting only milliseconds, the main methods used to capture them have been conventional imaging of analog or inhibited states, having extended lifetimes, or rapid (millisecond timescale) freezing of intermediates with subsequent observation by cryo-EM. We have developed a simpler method that fixes structure on the millisecond timescale. The procedure consists of briefly (milliseconds) exposing the macromolecular structure of interest on an EM grid to conditions that initiate the structural change, then immediately fixing with uranyl acetate or tannic acid. Specimens are then observed by negative staining. The key finding that validates this approach is our demonstration that uranyl acetate, and in some cases tannic acid, fixes protein molecular structure on the millisecond timescale. This is demonstrated by our observation that exposure of actin and myosin filaments to these fixatives for as little as 10 ms is sufficient to fully preserve them against changes that normally induce rapid and major alteration in their molecular structure. Fixation appears to stabilize both ionic and hydrophobic bonds. This approach should be of general utility for studying transient molecular changes in many systems.