Disulfide bond effects on protein stability: designed variants of Cucurbita maxima trypsin inhibitor-V

Attempts to increase protein stability by insertion of novel disulfide bonds have not always been successful. According to the two current models, cross-links enhance stability mainly through denatured state effects. We have investigated the effects of removal and addition of disulfide cross-links, protein flexibility in the vicinity of a cross-link, and disulfide loop size on the stability of Cucurbita maxima trypsin inhibitor-V (CMTI-V; 7 kD) by differential scanning calorimetry. CMTI-V offers the advantage of a large, flexible, and solvent-exposed loop not involved in extensive intra-molecular interactions. We have uncovered a negative correlation between retention time in hydrophobic column chromatography, a measure of protein hydrophobicity, and melting temperature (T(m)), an indicator of native state stabilization, for CMTI-V and its variants. In conjunction with the complete set of thermodynamic parameters of denaturation, this has led to the following deductions: (1) In the less stable, disulfide-removed C3S/C48S (Delta Delta G(d)(50 degrees C) = -4 kcal/mole; Delta T(m) = -22 degrees C), the native state is destabilized more than the denatured state; this also applies to the less-stable CMTI-V* (Delta Delta G(d)(50 degrees C) = -3 kcal/mole; Delta T(m) = -11 degrees C), in which the disulfide-containing loop is opened by specific hydrolysis of the Lys(44)-Asp(45) peptide bond; (2) In the less stable, disulfide-inserted E38C/W54C (Delta Delta G(d)(50 degrees C) = -1 kcal/mole; Delta T(m) = +2 degrees C), the denatured state is more stabilized than the native state; and (3) In the more stable, disulfide-engineered V42C/R52C (Delta Delta G(d)(50 degrees C) = +1 kcal/mole; Delta T(m) = +17 degrees C), the native state is more stabilized than the denatured state. These results show that a cross-link stabilizes both native and denatured states, and differential stabilization of the two states causes either loss or gain in protein stability. Removal of hydrogen bonds in the same flexible region of CMTI-V resulted in less destabilization despite larger changes in the enthalpy and entropy of denaturation. The effect of a cross-link on the denatured state of CMTI-V was estimated directly by means of a four-state thermodynamic cycle consisting of native and denatured states of CMTI-V and CMTI-V*. Overall, the results show that an enthalpy-entropy compensation accompanies disulfide bond effects and protein stabilization is profoundly modulated by altered hydrophobicity of both native and denatured states, altered flexibility near the cross-link, and residual structure in the denatured state.     

Substrate-induced activation of a trapped IMC-mediated protein folding intermediate

While several unfolded proteins acquire native structures through distinct folding intermediates, the physiological relevance and importance of such states in the folding kinetics remain controversial. The intramolecular chaperone (IMC) of subtilisin was used to trap a partially folded, stable crosslinked intermediate conformer (CLIC) through a disulfide bond between mutated IMC and subtilisin. The trapped CLIC contains non-native interactions. Here we show that CLIC can be induced into a catalytically active form by incubating it with small peptide substrates. The structure and catalytic properties of the activated crosslinked intermediate conformer (A-CLIC) differ from those of the fully folded enzyme in that A-CLIC lacks any endopeptidase activity toward a large protein substrate. Our results show that a disulfide-linked partially folded protein can be induced to acquire catalytic activity with a substrate specificity that is different from completely folded subtilisin. These results also suggest that protein folding intermediates may also participate in catalytic reactions.     

Synthesis and Biological Activity of Scyllatoxin-Based BH3 Domain Mimetics Containing Two Disulfide Linkages

The B cell lymphoma 2 (BCL2) proteins are a family of evolutionarily related proteins that act as positive or negative regulators of the intrinsic apoptosis pathway. Overexpression of anti-apoptotic BCL2 proteins in cells is associated with apoptotic resistance, which can result in cancerous phenotypes and pathogenic cell survival. Consequently, anti-apoptotic BCL2 proteins have attracted considerable interest as therapeutic targets. We recently reported the development of a novel class of synthetic protein based on scyllatoxin (ScTx) designed to mimic the helical BH3 interaction domain of the pro-apoptotic BCL2 protein Bax. These studies showed that the number and position of native disulfide linkages contained within the ScTx-Bax structure significantly influences the ability for these constructs to target anti-apoptotic BCL2 proteins in vitro. The goal of the present study is to investigate the contribution of two disulfide linkages in the folding and biological activity of ScTx-Bax proteins. Here, we report the full chemical synthesis of three ScTx-Bax sequence variants, each presenting two native disulfide linkages at different positions within the folded structure. It was observed that two disulfide linkages were sufficient to fold ScTx-Bax proteins into native-like architectures reminiscent of wild-type ScTx. Furthermore, we show that select (bis)disulfide ScTx-Bax variants can target Bcl-2 (proper) in vitro and that the position of the disulfide bonds significantly influences binding affinity. Despite exhibiting only modest binding to Bcl-2, the successful synthesis of ScTx-Bax proteins containing two disulfide linkages represents a viable route to ScTx-based BH3 domain mimetics that preserve native-like conformations. Finally, structural models of ScTx-Bax proteins in complex with Bcl-2 indicate that these helical mimetics bind in similar configurations as wild-type Bax BH3 domains. Taken together, these results suggest that ScTx-Bax proteins may serve as potent lead compounds that expand the repertoire of “druggable” protein–protein interactions.     

Insulin-like growth factors I and II are unable to form and maintain their native disulfides under in vivo redox conditions

Insulin-like growth factor (IGF) I does not quantitatively form its three native disulfide bonds in the presence of 10 mM reduced and 1 mM oxidized glutathione in vitro [Hober, S. et al. (1992) Biochemistry 31, 1749-1756]. In this paper, we show (i) that both IGF-I and IGF-II are unable to form and maintain their native disulfide bonds at redox conditions that are similar to the situation in the secretory vesicles in vivo and (ii) that the presence of protein disulfide isomerase does not overcome this problem. The results indicate that the previously described thermodynamic disulfide exchange folding problem of IGF-I in vitro is also present in vivo. Speculatively, we suggest that the thermodynamic disulfide exchange properties of IGF-I and II are biologically significant for inactivation of the unbound growth factors by disulfide exchange reactions to generate variants destined for rapid clearance.     

Nonnative interactions between cysteines direct productive assembly of P22 tailspike protein

Nonnative disulfide bond formation can play a critical role in the assembly of disulfide bonded proteins. During the folding and assembly of the P22 tailspike protein, nonnative disulfide bonds form both in vivo and in vitro. However, the mechanism and identity of cysteine disulfide pairs remains elusive, particularly for P22 tailspike, which contains no disulfide bonds in its native, functional form. Understanding the interactions between cysteine residues is important for developing a mechanistic model for the role of nonnative cysteines in P22 tailspike assembly. Prior in vivo studies have suggested that cysteines 496, 613, and 635 are the most likely site for sulfhydryl reactivity. Here we demonstrate that these three cysteines are critical for efficient assembly of tailspike trimers, and that interactions between cysteine pairs lead to productive assembly of native tailspike.     

Optimization of the electrostatic interactions in proteins of different functional and folding type

The 3-dimensional optimization of the electrostatic interactions between the charged amino acid residues was studied by Monte Carlo simulations on an extended representative set of 141 protein structures with known atomic coordinates. The proteins were classified by different functional and structural criteria, and the optimization of the electrostatic interactions was analyzed. The optimization parameters were obtained by comparison of the contribution of charge-charge interactions to the free energy of the native protein structures and for a large number of randomly distributed charge constellations obtained by the Monte Carlo technique. On the basis of the results obtained, one can conclude that the charge-charge interactions are better optimized in the enzymes than in the proteins without enzymatic functions. Proteins that belong to the mixed αβ folding type are electrostatically better optimized than pure α-helical or β-strand structures. Proteins that are stabilized by disulfide bonds show a lower degree of electrostatic optimization. The electrostatic interactions in a native protein are effectively optimized by rejection of the conformers that lead to repulsive charge-charge interactions. Particularly, the rejection of the repulsive contacts seems to be a major goal in the protein folding process. The dependence of the optimization parameters on the choice of the potential function was tested. The majority of the potential functions gave practically identical results.     

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。     

Native disulfide bonds greatly accelerate secondary structure formation in the folding of lysozyme.

To assess the respective roles of local and long-range interactions during protein folding, the influence of the native disulfide bonds on the early formation of secondary structure was investigated using continuous-flow circular dichroism. Within the first 4 ms of folding, lysozyme with intact disulfide bonds already had a far-UV CD spectrum reflecting large amounts of secondary structure. Conversely, reduced lysozyme remained essentially unfolded at this early folding time. Thus, native disulfide bonds not only stabilize the cfinal conformation of lysozyme but also provide, in early folding intermediates, the necessary stabilization that favors the formation of secondary structure.     

SMA-PAGE: A new method to examine complexes of membrane proteins using SMALP nano-encapsulation and native gel electrophoresis

Most membrane proteins function through interactions with other proteins in the phospholipid bilayer, the cytosol or the extracellular milieu. Understanding the molecular basis of these interactions is key to understanding membrane protein function and dysfunction. Here we demonstrate for the first time how a nano-encapsulation method based on styrene maleic acid lipid particles (SMALPs) can be used in combination with native gel electrophoresis to separate membrane protein complexes in their native state. Using four model proteins, we show that this separation method provides an excellent measure of protein quaternary structure, and that the lipid environment surrounding the protein(s) can be probed using mass spectrometry. We also show that the method is complementary to immunoblotting. Finally we show that intact membrane protein-SMALPs extracted from a band on a gel could be visualised using electron microscopy (EM). Taken together these results provide a novel and elegant method for investigating membrane protein complexes in a native state.     

Role of intramolecular disulfides in stability and structure of a noncovalent homodimer

The importance of intramolecular disulfides in a noncovalent dimeric protein interleukin-8 (IL-8) has been studied by replacing cysteines in each of the two disulfide pairs with alpha-aminobutyric acid (CH(2)-SH --> CH(2)-CH(3)). Both disulfide mutants are less stable and exist as molten globules in the monomeric state. Interestingly, both mutants dimerize, though with slightly lower affinities compared to the native protein. NMR studies suggest a molten globule-like structure also in the dimeric state. Structures, sequence analysis, and mutagenesis studies have shown that the conserved hydrophobic residues are packed against each other in the protein core and that H bonding and van der Waals interactions stabilize the dimer interface. Deleting either disulfide in IL-8 results in substantial loss in receptor activity, indicating that both disulfides are critical for function in the folded protein. These data together suggest that the packing interactions of the hydrophobic core determine IL-8 monomer fold, that disulfides play only a marginal role in dimer formation, and that the stability imparted by the disulfides is intimately coupled to fold and function.     

Mucin granule intraluminal organization in living mucous/goblet cells. Roles of protein post-translational modifications and secretion

Recent studies suggest that the mucin granule lumen consists of a matrix meshwork embedded in a fluid phase. Secretory products can both diffuse, although very slowly, through the meshwork pores and interact noncovalently with the matrix. Using a green fluorescent protein-mucin fusion protein (SHGFP-MUC5AC/CK) as a FRAP (fluorescence recovery after photobleaching) probe, we have assessed in living mucous cells the relative importance of different protein post-translational modifications on the intragranular organization. Long term inhibition of mucin-type O-glycosylation, sialylation, or sulfation altered SHGFP-MUC5AC/CK characteristic diffusion time (t(1/2)), whereas all but sulfation diminished its mobile fraction. Reduction of protein disulfide bonds with tris(hydroxypropyl)phosphine resulted in virtually complete immobilization of the SHGFP-MUC5AC/CK intragranular pool. However, when activity of the vacuolar H+-ATPase was also inhibited, disulfide reduction decreased SHGFP-MUC5AC/CK t((1/2)) while diminishing its intraluminal concentration. Similar FRAP profiles were observed in granules that remained in the cells after the addition of a mucin secretagogue. Taken together these results suggest that: (a) the relative content of O-glycans and intragranular anionic groups is crucial for protein diffusion through the intragranular meshwork; (b) protein-protein, rather than carbohydrate-mediated, interactions are responsible for binding of SHGFP-MUC5AC/CK to the immobile fraction, although the degree of matrix O-glycosylation and sialylation affects such interactions; (c) intragranular organization does not depend on covalent multimerization of mucins or the presence of native disulfide bonds in the intragranular mucin/proteins, but rather on specific protein-mediated interactions that are important during the early stages of mucin matrix condensation; (d) alterations of the intragranular matrix precede granule discharge, which can be partial and, accordingly, does not necessarily involve the disappearance of the granule.     

Non-redox protein interactions in the thioredoxin activation of chloroplast enzymes.

Thioredoxin derivatives lacking SH groups such as S,S'-dicarboxymethyl-, dicarboxamidomethyl-thioredoxin and cysteine----serine mutant protein are capable of activating chloroplast NADP malate dehydrogenase and fructose-bisphosphatase when added to enzyme assays together with suboptimal amounts of native thioredoxin. The modified thioredoxins alone are inactive. These findings indicate that protein-protein interactions play a significant role in addition to disulfide/thiol exchange reactions in the light-driven regulation of plant enzymes by the various plant thioredoxins.     

Shifting the competition between the intramolecular Reshuffling reaction and the direct oxidation reaction during the oxidative folding of kinetically trapped disulfide-insecure intermediates

The oxidative folding pathway(s) of single-domain proteins can be characterized by the existence, stability, and structural nature of the intermediates that populate the regeneration pathway. Structured intermediates can be disulfide-secure in that they are able to protect their existing (native) disulfide bonds from SH/SS reshuffling and reduction reactions, and thereby form the native protein directly, i.e., by oxidation of their exposed (or locally exposable) thiols. Alternatively, they can be disulfide-insecure, usually requiring global unfolding to expose their free thiols. However, such an unfolding event also exposes the existing native disulfide bonds. Thus, the subsequent oxidation reaction to form the native protein in a disulfide-insecure intermediate competes with the intramolecular attack by the thiols of the macromolecule on its own native disulfide bonds, resulting in a large population of intermediates that are reshuffled instead of being oxidized. Under stabilizing conditions, disulfide-insecure species become long-lived kinetically trapped intermediates that slowly and only indirectly convert to the native protein through reshuffling reactions. In this study, trans-[Pt(en)(2)Cl(2)](2+), a strong oxidizing agent which has not traditionally been used in oxidative folding, was applied to shift the competition between reshuffling and oxidation reactions in des [58-110] and des [26-84], two long-lived disulfide-insecure intermediates of RNase A, and oxidize them directly under stable conditions to form the native protein. Such a successful direct conversion of kinetically trapped intermediates to the native molecule by trans-[Pt(en)(2)Cl(2)](2+) may be helpful in facilitating the oxidative folding processes of multi-disulfide-containing proteins in general.     

Determining the roles of different chain fragments in recognition of immunoglobulin fold

We examine sequence-to-structure specificity of beta-structural fragments of immunoglobulin domains. The structure specificity of separate chain fragments is estimated by computing the Z-score values in recognition of the native structure in gapless threading tests. To improve the accuracy of our calculations we use energy averaging over diverse homologs of immunoglobulin domains. We show that the interactions between residues of beta-structure are more determinant in recognition of the native structure than the interactions within the whole chain molecule. This result distinguishes immunoglobulins from more typical proteins where the interactions between residues of the whole chain normally recognize the native fold more accurately than interactions between the residues of the secondary structure residues alone [Reva,B. and Topiol,S. (2000) BIOCOMPUTING: Proceedings of the Pacific Symposium. World Scientific Publishing Co., pp. 168-178]. We also find that the predominant contributions of the secondary structure are produced by the four central beta-strands that form the core of the molecule. The results of this study allow us through quantitative means to understand the architecture of immunoglobulin molecules. Comparing the fold recognition data for different chain fragments one can say that beta-strands form a rigid frame for immunoglobulin molecules, whereas loops, with no structural role, can develop a broad variety of binding specificities. It is well known that protein function is determined by specific portions of a protein chain. This study suggests that the whole protein structure can be predominantly determined by a few fragments of chain which form the structural framework of the molecule. This idea may help in better understanding the mechanisms of protein evolution: strengthening a protein structure in the key framework-forming regions allows mutations and flexibility in other chain regions.     

Initial disulfide formation steps in the folding of an omega-conotoxin

To determine whether the native disulfides of omega-conotoxins are preferentially stabilized early in the folding of these small proteins, the rates and equilibria for disulfide formation were measured for three analogues of omega-conotoxin MVIIA. In each analogue, one of the three pairs of disulfide-bonded Cys residues was replaced with Ala residues, leaving four Cys residues that can form six intermediates with one disulfide and three species with two disulfides. For each analogue, all of the disulfide-bonded species were identified, and the equilibrium constants for forming the individual species via exchange with oxidized and reduced glutathione were measured. These equilibrium constants represent effective concentrations of the Cys thiols and ranged from 0.01 to 0.4 M in the fully reduced protein. There was little or no preference for forming the native disulfides, and the equilibria for forming the first and second disulfides decreased only slightly upon the addition of 8 M urea. The data for the four-Cys analogues, together with equilibrium data for the six-Cys form, were also used to estimate effective concentrations for forming a third disulfide once two native disulfides are present. These effective concentrations were approximately 100 and 10 M in the presence of 0 and 8 M urea, respectively. The results indicate that there is little or no preferential formation of native interactions in the folding of these molecules until two disulfides have formed, after which there is a high degree of cooperativity among the native interactions.     

Interchain disulfide bond formation in types I and II procollagen. Evidence for a protein disulfide isomerase catalyzing bond formation

The assembly of reduced pro-alpha chains of type I and type II procollagen into the native triple-helical molecule was examined in vitro in the presence and absence of pure protein disulfide isomerase. The data clearly indicates that protein disulfide isomerase is able to accelerate the formation of native interchain disulfide bonds in these procollagens. It takes about 6 min after disulfide bonding before triple-helical molecules exist, while the time required to produce triple-helical type I procollagen in the presence of protein disulfide isomerase is 9.4 min and that for type II procollagen 17.2 min. These values agree with those obtained for type I and II procollagen in vivo suggesting that protein disulfide isomerase is also an enzyme catalyzing interchain disulfide bond formation in procollagen in vivo. The formation of native disulfide bonds can proceed without any enzyme catalysis but then requires the presence of reduced and oxidized glutathione. Bonding is rather slow in such a case, however, resulting in a delay in the formation of the triple helix.     

Synthesis and biological properties of carba-analogs of heat-stable enterotoxin (ST) produced by enterotoxigenic Escherichia coli.

Analogs of a heat-stable enterotoxin (ST) that have a CH2-S linkage instead of an S-S linkage in the molecule were synthesized by conventional methods. The synthetic peptides showed toxicity, assayed as induction of fluid secretion in suckling mice, although their toxicities were hundredth that of native ST. This finding implies that ST is not recognized by its receptor protein through an exchange reaction between its disulfide linkages and thiol-groups of its receptor protein(s), but through hydrophobic or electrostatic interactions.     

The multisubunit structure of synaptophysin. Relationship between disulfide bonding and homo-oligomerization

Synaptophysin, a major membrane protein of synaptic vesicles, contains four transmembrane regions and two intravesicular loops. Synaptophysin monomers associate into homopolymers that have the potential to form channels in the synaptic vesicle membrane. Here we show that in native synaptophysin, homopolymers are linked by noncovalent forces. The molecule contains unstable intramolecular disulfide bonds that undergo disulfide exchange during solubilization, thereby covalently cross-linking neighboring synaptophysin molecules. The locations of the intramolecular disulfide bonds in synaptophysin were determined, revealing that each of the two intravesicular loops of synaptophysin is circularized by a single disulfide bond. Cross-linking of synaptophysin by disulfide bonds can be triggered in synaptic vesicles and in intact cells by a cycle of reduction and oxidation, suggesting that native synaptophysin is a homomultimer in situ. In addition, chemical cross-linking of native synaptophysin demonstrates that a low molecular weight protein is specifically associated with synaptophysin complexes and is lost upon reduction of the intramolecular disulfide bonds. These data suggest that native synaptophysin forms a noncovalent homomultimeric complex whose structure and interaction with other proteins are dependent on the integrity of its intramolecular disulfide bonds and phospholipid environment.     

The fusion-controlling disulfide bond isomerase in retrovirus Env is triggered by protein destabilization

The membrane fusion function of murine leukemia virus (MLV) is carried by the Env protein. This protein is composed of three SU-TM subunit complexes. The fusion activity is loaded into the transmembrane TM subunit and controlled by the peripheral, receptor-binding SU subunit. It is assumed that TM adopts a metastable conformation in the native Env and that fusion activation involves the folding of TM into a stable form. Activation is suppressed by the associated SU and triggered by its dissociation, which follows receptor binding. Recently we showed that the two subunits are disulfide linked and that SU dissociation and triggering of the fusion function are caused by a switch of the intersubunit disulfide into an intrasubunit disulfide isomer using an isomerization-active CWLC motif in SU (M. Wallin, M. Ekstrom, and H. Garoff, EMBO J. 23:54-65, 2004). In the present work we address how the SU disulfide isomerase is activated. Using Moloney MLV, we show that isomerization of the SU-TM disulfide bond can be triggered by heat, urea, or guanidinium hydrochloride. Such protein perturbation treatments also significantly increase the kinetics and efficiency of viral fusion. The threshold conditions for the effects on isomerization and fusion are virtually the same. This finding indicates that destabilization of interactions in the SU oligomer induces the disulfide bond isomerase and the subsequent activation of the fusion function in TM.