Structure and heterogeneity of the one- and two-disulfide folding intermediates of tick anticoagulant peptide

Tick anticoagulant peptide (TAP) is a factor Xa-specific inhibitor and is structurally homologous to bovine pancreatic trypsin inhibitor (BPTI). The fully reduced TAP refolds spontaneously to form the native structure under a wide variation of redox buffers. The folding intermediates of TAP consist of at least 22 fractions of one-disulfide, two-disulfide, and three-disulfide scrambled isomers. Three species of well-populated one- and two-disulfide intermediates were isolated and structurally characterized. The predominant one-disulfide species contains TAP-(Cys33—Cys55). Two major two-disulfide isomers were TAP-(Cys33—Cys55, Cys15—Cys39) and TAP-(Cys33—Cys55, Cys5—Cys39). Both Cys33—Cys55 and Cys15—Cys39 are native disulfides of TAP. These three species are structural counterparts of BPTI-(Cys30—Cys51), BPTI-(Cys30—Cys51, Cys14—Cys38), and BPTI-(Cys30—Cys51,Cys5—Cys38), which have been shown to be the major intermediates of BPTI folding. In addition, time-course-trapped folding intermediates of TAP, consisting of about 47% one-disulfide species and 30% two-disulfide species, were collectively digested with thermolysin, and fragmented peptides were analyzed by Edman sequencing and mass spectrometry in order to characterize the disulfide-containing peptides. Among the 15 possible single-disulfide pairings of TAP, 10 (2 native and 8 nonnative) were found as structural components of its one- and two-disulfide folding intermediates. The results demonstrate that the major folding intermediates of TAP bear structural homology to those of BPTI. However, the folding pathway of TAP differs from that of BPTI by (a) a higher degree of heterogeneity of one- and two-disulfide intermediates and (b) the presence of three-disulfide scrambled isomers as folding intermediates. Mechanism(s) that may account for these diversities are proposed and discussed.     

Folding in vitro of bovine pancreatic trypsin inhibitor in the presence of proteins of the endoplasmic reticulum.

The rates of folding and disulfide bond formation in reduced BPTI were measured in vitro in the presence and absence of total protein from the endoplasmic reticulum. The rates were increased substantially by the endoplasmic reticulum proteins, but only to the extent expected from the known content and activity of protein-disulfide-isomerase. No effects of added ATP or Ca2+ were observed, even though protein-disulfide-isomerase binds Ca2+ tightly.     

Probing protein folding and stability using disulfide bonds

Disulfide bonds are required to stabilize the folded conformations of many proteins. The rates and equilibria of processes involved in disulfide bond formation and breakage can be manipulated experimentally and can be used to obtain important information about protein folding and stability. A number of experimental procedures for studying these processes, and approaches to interpreting the resulting data, are described here.     

Further experimental studies of the disulfide folding transition of ribonuclease A

Two very different mechanisms of folding have been proposed from experimental studies of disulfide formation in reduced ribonuclease A. (1) A pathway in which the rate-limiting step separates fully folded protein from all other disulfide intermediates and occurs solely in three-disulfide intermediates. (2) A multiple pathway mechanism with different rate-limiting steps for each pathway. The various rate-limiting steps involve disulfide breakage, formation, and rearrangement in intermediates with one, two, three, and four protein disulfides. To distinguish between these two mechanisms, we have carried out further studies of both unfolding and refolding. Refolding of reduced ribonuclease A requires three-disulfide intermediates to accumulate; negligible refolding occurs when only the nearly random one- and two-disulfide intermediate species are populated. Therefore, no rate-limiting steps of the type postulated in mechanism (2) occur in intermediates with one and two protein disulfides. Unfolding and disulfide reduction is an all-or-none process; no disulfide intermediates accumulate to detectable levels or precede the rate-limiting step. Mechanism (2) requires that such intermediates precede the rate-limiting step and accumulate to substantial levels. The different proposals were shown not to result from the use of different solution conditions or disulfide reagents; the two sets of data are not inconsistent. Instead, the inappropriate mechanism (2) resulted from an incorrect kinetic analysis and misinterpretation of the kinetics of disulfide formation and breakage.     

A study of intermediates involved in the folding pathway for recombinant human macrophage colony-stimulating factor (M-CSF): evidence for two distinct folding pathways.

The folding pathway for a 150-amino acid recombinant form of the dimeric cytokine human macrophage colony-stimulating factor (M-CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a non-native dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer. The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0. The blocked intermediates are stable under conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges. Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M-CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell-based assay. The results presented here strongly suggest that M-CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates.     

Intradomain disulfide bonds impede formation of the alternatively folded state of antibody chains

Antibodies undergo significant conformational changes upon acidification, leading to the formation of an alternatively folded state. Here, we analyzed the conformation of MAK 33 Fab and its light chain at acidic pH, both in the reduced and oxidized form. At acidic pH, the proteins exhibited a highly structured, but non-native conformation, corresponding to the alternatively folded state, previously described for the intact antibody. However, the requirements to form this alternative structure were different for the oxidized and reduced protein. Whereas in the oxidized form of the immunoglobulin light chain the alternatively folded state could only be detected at pH<1.4, the reduced light chain already adopted this structure at pH 2. Thermal denaturation measurements revealed that, surprisingly, the alternatively folded state of the reduced light chain was more stable than that of the oxidized protein at pH 1.4. This indicates that the intradomain disulfide bonds, which stabilize the native state of antibody domains, impede the formation of the alternatively folded state.     

Molecular basis of cooperativity in protein folding IV. Core: A general cooperative folding model

The cooperative nature of the protein folding process is independent of the characteristic fold and the specific secondary structure attributes of a globular protein. A general folding/unfolding model should, therefore, be based upon structural features that transcend the peculiarities of α-helices, β-sheets, and other structural motifs found in proteins. The studies presented in this paper suggest that a single structural characteristic common to all globular proteins is essential for cooperative folding. The formation of a partly folded state from the native state results in the exposure to solvent of two distinct regions: (1) the portions of the protein that are unfolded; and (2) the “complementary surfaces,” located in the regions of the protein that remain folded. The cooperative character of the folding/unfolding transition is determined largely by the energetics of exposing complementary surface regions to the solvent. By definition, complementary regions are present only in partly folded states; they are absent from the native and unfolded states. An unfavorable free energy lowers the probability of partly folded states and increases the cooperativity of the transition. In this paper we present a mathematical formulation of this behavior and develop a general cooperative folding/unfolding model, termed the “complementary region” (CORE) model. This model successfully reproduces the main properties of folding/unfolding transitions without limiting the number of partly folded states accessible to the protein, thereby permitting a systematic examination of the structural and solvent conditions under which intermediates become populated. It is shown that the CORE model predicts two-state folding/unfolding behavior, even though the two-state character is not assumed in the model. © 1993 Wiley-Liss, Inc.     

Folding/Unfolding Kinetics of Apomyoglobin

The equilibrium and kinetic folding/unfolding of apomyoglobin (ApoMb) were studied at pH 6.2, 11 °C by recording tryptophan fluorescence. The equilibrium unfolding of ApoMb in the presence of urea was shown to involve accumulation of an intermediate state, which had a higher fluorescence intensity as compared with the native and unfolded states. The folding proceeded through two kinetic phases, a rapid transition from the unfolded to the intermediate state and a slow transition from the intermediate to the native state. The accumulation of the kinetic intermediate state was observed in a wide range of urea concentrations. The intermediate was detected even in the region corresponding to the unfolding limb of the chevron plot. Urea concentration dependence was obtained for the observed folding/unfolding rate. The shape of the dependence was compared with that of two-state proteins characterized by a direct transition from the unfolded to the native state.     

Fast folding of a prototypic polypeptide: the immunoglobulin binding domain of streptococcal protein G.

The folding of the small (56 residues) highly stable B1 immunoglobulin binding domain (GB1) of streptococcal protein G has been investigated by quenched-flow deuterium-hydrogen exchange. This system represents a paradigm for the study of protein folding because it exhibits no complicating features superimposed upon the intrinsic properties of the polypeptide chain. Collapse to a semicompact state exhibiting partial order, reflected in protection factors for ND-NH exchange up to 10-fold higher than that expected for a random coil, occurs within the dead time (< or = 1 ms) of the quenched flow apparatus. This is followed by the formation of the fully native state, as monitored by the fractional proton occupancy of 26 backbone amide groups spread throughout the protein, in a single rapid concerted step with a half-life of 5.2 ms at 5 degrees C.     

Lipase maturation factor 1 affects redox homeostasis in the endoplasmic reticulum

Lipoprotein lipase (LPL) is a secreted lipase that clears triglycerides from the blood. Proper LPL folding and exit from the endoplasmic reticulum (ER) require lipase maturation factor 1 (LMF1), an ER‐resident transmembrane protein, but the mechanism involved is unknown. We used proteomics to identify LMF1‐binding partners necessary for LPL secretion in HEK293 cells and found these to include oxidoreductases and lectin chaperones, suggesting that LMF1 facilitates the formation of LPL's five disulfide bonds. In accordance with this role, we found that LPL aggregates in LMF1‐deficient cells due to the formation of incorrect intermolecular disulfide bonds. Cells lacking LMF1 were hypersensitive to depletion of glutathione, but not DTT treatment, suggesting that LMF1 helps reduce the ER. Accordingly, we found that loss of LMF1 results in a more oxidized ER. Our data show that LMF1 has a broader role than simply folding lipases, and we identified fibronectin and the low‐density lipoprotein receptor (LDLR) as novel LMF1 clients that contain multiple, non‐sequential disulfide bonds. We conclude that LMF1 is needed for secretion of some ER client proteins that require reduction of non‐native disulfides during their folding.     

GroEL Can Unfold Late Intermediates Populated on the Folding Pathways of Monellin

The modulation of the folding mechanism of the small protein single-chain monellin (MNEI) by the Escherichia coli chaperone GroEL has been studied. In the absence of the chaperone, the folding of monellin occurs via three parallel routes. When folding is initiated in the presence of a saturating concentration of GroEL, only 50-60% of monellin molecules fold completely. The remaining 40-50% of the monellin molecules remain bound to the GroEL and are released only upon addition of ATP. It is shown that the basic folding mechanism of monellin is not altered by the presence of GroEL, but that it occurs via only one of the three available routes when folding is initiated in the presence of saturating concentrations of GroEL. Two pathways become nonoperational because GroEL binds very tightly to early intermediates that populate these pathways in a manner that makes the GroEL-bound intermediates incompetent to fold. This accounts for the monellin molecules that remain GroEL-bound at the end of the folding reaction. The third pathway remains operational because the GroEL-bound early intermediate on this pathway is folding-competent, suggesting that this early intermediate binds to GroEL in a manner that is different from that of the binding of the early intermediates on the other two pathways. It appears, therefore, that the same protein can bind GroEL in more than one way. The modulation of the folding energy landscape of monellin by GroEL occurs because GroEL binds folding intermediates on parallel folding pathways, in different ways, and with different affinities. Moreover, when GroEL is added to refolding monellin at different times after commencement of refolding, the unfolding of two late kinetic intermediates on two of the three folding pathways can be observed. It appears that the unfolding of late folding intermediates is enabled by a thermodynamic coupling mechanism, wherein GroEL binds more tightly to an early intermediate than to a late intermediate on a folding pathway, with preferential binding energy being larger than the stability of the late intermediate. Hence, it is shown that GroEL can inadvertently and passively cause, through its ability to bind different folding intermediates differentially, the unfolding of late productive intermediates on folding pathways, and that its unfolding action is not restricted solely to misfolded or kinetically trapped intermediates.