Unfolding and refolding of cytochrome c driven by the interaction with lipid micelles

Binding of native cyt c to L-PG micelles leads to a partially unfolded conformation of cyt c. This micelle-bound state has no stable tertiary structure, but remains as alpha-helical as native cyt c in solution. In contrast, binding of the acid-unfolded cyt c to L-PG micelles induces folding of the polypeptide, resulting in a similar helical state to that originated from the binding of native cyt c to L-PG micelles. Far-ultraviolet (UV) circular dichroism (CD) spectra showed that this common micelle-associated helical state (HL) has a native-like alpha-helix content, but is highly expanded without a tightly packed hydrophobic core, as revealed by tryptophan fluorescence, near-UV, and Soret CD spectroscopy. The kinetics of the interaction of native and acid-unfolded cyt c was investigated by stopped-flow tryptophan fluorescence. Formation of H(L) from the native state requires the disruption of the tightly packed hydrophobic core in the native protein. This micelle-induced unfolding of cyt c occurs at a rate approximately 0.1 s(-1), which is remarkably faster in the lipid environment compared with the expected rate of unfolding in solution. Refolding of acid-unfolded cyt c with L-PG micelles involves an early highly helical collapsed state formed during the burst phase (<3 ms), and the observed main kinetic event reports on the opening of this early compact intermediate prior to insertion into the lipid micelle.     

Structure and dynamics of lipid-associated states of apocytochrome c.

Apocytochrome c (apocyt c), which in aqueous solution is largely unstructured, acquires an alpha-helical conformation upon association with lipid membranes. The extent of alpha-helix induced in apocyt c is lipid-dependent and this folding process is driven by both electrostatic and hydrophobic lipid-protein interactions. The structural and dynamic properties of apocyt c in lipid membranes were investigated by attenuated total reflection Fourier transform infrared spectroscopy combined with amide H-D exchange kinetics. Apocyt c acquires a higher content of alpha-helical structure with negatively charged membranes than with zwitterionic ones. For all membranes studied here, the helices of these partially folded states of apocyt c have a preferential orientation perpendicular to the plane of the lipid membrane. The H-D exchange revealed that a small fraction of amide protons of apocyt c, possibly associated with a stable folded domain protected by the lipid, remained protected from exchange over 20 min. However, a large fraction of amide protons exchanged in less than 20 min, indicating that the helical states of apocyt c in lipid membranes are very dynamic.     

Stepwise formation of alpha-helices during cytochrome c folding

Two models have been proposed to describe the folding pathways of proteins. The framework model assumes the initial formation of the secondary structures whereas the hydrophobic collapse model supposes their formation after the collapse of backbone structures. To differentiate between these models for real proteins, we have developed a novel CD spectrometer that enables us to observe the submillisecond time frame of protein folding and have characterized the timing of secondary structure formation in the folding process of cytochrome c (cyt c). We found that approximately 20% of the native helical content was organized in the first phase of folding, which is completed within milliseconds. Furthermore, we suggest the presence of a second intermediate, which has alpha-helical content resembling that of the molten globule state. Our results indicate that many of the alpha-helices are organized after collapse in the folding mechanism of cyt c.     

Folding of apocytochrome c in lipid micelles: formation of alpha-helix precedes membrane insertion.

Apocytochrome c, which in aqueous solution is largely unstructured, acquires a highly alpha-helical structure upon interaction with lipid. The alpha-helix content induced in apocytochrome c depends on the lipid system, and this folding process is driven by both electrostatic and hydrophobic lipid-protein interactions. The folding kinetic mechanism of apocytochrome c induced by zwitterionic micelles of lysophosphatidylcholine (L-PC), predominantly driven by hydrophobic lipid-protein interactions, was investigated by fluorescence stopped-flow measurements of Trp 59 and fluorescein-phosphatidylethanolamine-(FPE) labeled micelles, in combination with stopped-flow far-UV circular dichroism. It was found that formation of the alpha-helical structure of apocytochrome c precedes membrane insertion. The unfolded state in solution (U(W)) binds to the micelle surface in a helical conformation (I(S)) and is followed by insertion into the lipid micelle, i.e., formation of the final helical state H(L). Binding of apocytochrome c to the lipid micelle (U(W) --> I(S)) is concurrent with formation of a large fraction (75-100%, depending on lipid concentration) of the alpha-helical structure of the final lipid-inserted state H(L). The highly helical intermediate I(S) is formed on the time scale of 3-12 ms, depending on lipid concentration, and inserts into the lipid micelle (I(S) --> H(L)) in the time range of approximately 200 ms to >1 s, depending on lipid-to-protein ratio. The final lipid-inserted helical state H(L) in L-PC micelles has an alpha-helix content approximately 65% of that of cytochrome c in solution and has no compact stable tertiary structure as revealed by circular dichroism results.     

Structural transformation of apocytochrome c induced by alternating copolymers of maleic acid and alkene

Apocytochrome c interacts with two copolymers: poly(isobutylene-alt-maleic acid) (PIMA) and poly(1-tetradecene-alt-maleic acid) (PTMA). The interaction leads to apocytochrome c, a conformational change from random coil to alpha-helical structure. The alpha-helix content is influenced by the copolymer concentration, the length of alkyl chain of the copolymers, and pH of the medium. The electrostatic attraction between the copolymer and protein is an indispensable factor for the folding of the protein at acid pH. The hydrophobic interaction is an important factor over the entire pH range, especially when both the copolymer and protein carry negative charges at alkaline pH. The electrostatic and hydrophobic attractions between the copolymer and protein exclude water molecules, promoting the formation of hydrogen bonds within the helical structure. On the other hand, the hydrogen bonds formed between the ionized carboxyl of the copolymer and the amide of the protein partly restrain the formation of hydrogen bonds within the helical structure when the copolymer concentration is higher at pH 6.5 and 10.5.     

Brownian dynamics study of cytochrome f interactions with cytochrome c6 and plastocyanin in Chlamydomonas reinhardtii plastocyanin, and cytochrome c6 mutants

Using Brownian dynamics simulations, all of the charged residues in Chlamydomonas reinhardtii cytochrome c(6) (cyt c(6)) and plastocyanin (PC) were mutated to alanine and their interactions with cytochrome f (cyt f) were modeled. Systematic mutation of charged residues on both PC and cyt c(6) confirmed that electrostatic interactions (at least in vitro) play an important role in bringing these proteins sufficiently close to cyt f to allow hydrophobic and van der Waals interactions to form the final electron transfer-active complex. The charged residue mutants on PC and cyt c(6) displayed similar inhibition classes. Our results indicate a difference between the two acidic clusters on PC. Mutations D44A and E43A of the lower cluster showed greater inhibition than do any of the mutations of the upper cluster residues. Replacement of acidic residues on cyt c(6) that correspond to the PC's lower cluster, particularly E70 and E69, was observed to be more inhibitory than those corresponding to the upper cluster. In PC residues D42, E43, D44, D53, D59, D61, and E85, and in cyt c(6) residues D2, E54, K57, D65, R66, E70, E71, and the heme had significant electrostatic contacts with cyt f charged residues. PC and cyt c(6) showed different binding sites and orientations on cyt f. As there are no experimental cyt c(6) mutation data available for algae, our results could serve as a good guide for future experimental work on this protein. The comparison between computational values and the available experimental data (for PC-cyt f interactions) showed overall good agreement, which supports the predictive power of Brownian dynamics simulations in mutagenesis studies.     

Involvement of electrostatic interactions in the mechanism of peptide folding induced by sodium dodecyl sulfate binding

Sodium dodecyl sulfate (SDS) has consistently been shown to induce secondary structure, particularly alpha-helices, in polypeptides, and is commonly used to model membrane and other hydrophobic environments. However, the precise mechanism by which SDS induces these conformational changes remains unclear. To examine the role of electrostatic interactions in this mechanism, we have designed two hydrophilic, charged amphipathic alpha-helical peptides, one basic (QAPAYKKAAKKLAES) and the other acidic (QAPAYEEAAEELAKS), and their structures were studied by CD and NMR. The design of the peptides is based on the sequence of the segment of residues 56-70 of human platelet factor 4 [PF4(56-70), QAPLYKKIIKKLLES]. Both peptides were unstructured in water, and in the presence of neutral, zwitterionic, or cationic detergents. However, in SDS at neutral pH, the basic peptide folded into an alpha-helix. By contrast, the pH needed to be lowered to 1.8 before alpha-helix formation was observed for the acidic peptide. Strong, attractive electrostatic interactions, between the anionic groups of SDS and the cationic groups of the lysines, appeared to be necessary to initiate the folding of the basic peptide. NMR analysis showed that the basic peptide was fully embedded in SDS-peptide micelles, and that its three-dimensional alpha-helical structure could be superimposed on that of the native structure of PF4(56-70). These results enabled us to propose a working model of the basic peptide-SDS complex, and a mechanism for SDS-induced alpha-helical folding. This study demonstrates that, while the folding of peptides is mostly driven by hydrophobic effects, electrostatic interactions play a significant role in the formation and the stabilization of SDS-induced structure.     

Folding of apocytochrome c induced by the interaction with negatively charged lipid micelles proceeds via a collapsed intermediate state

Unfolded apocytochrome c acquires an alpha-helical conformation upon interaction with lipid. Folding kinetic results below and above the lipid's CMC, together with energy transfer measurements of lipid bound states, and salt-induced compact states in solution, show that the folding transition of apocytochrome c from the unfolded state in solution to a lipid-inserted helical conformation proceeds via a collapsed intermediate state (I(C)). This initial compact state is driven by a hydrophobic collapse of the polypeptide chain in the absence of the heme group and may represent a heme-free analogue of an early compact intermediate detected on the folding pathway of cytochrome c in solution. Insertion into the lipid phase occurs via an unfolding step of I(C) through a more extended state associated with the membrane surface (I(S)). While I(C) appears to be as compact as salt-induced compact states in solution with substantial alpha-helix content, the final lipid-inserted state (Hmic) is as compact as the unfolded state in solution at pH 5 and has an alpha-helix content which resembles that of native cytochrome c.     

Brownian dynamics simulations of the interaction of Chlamydomonas cytochrome f with plastocyanin and cytochrome c6

The interaction of Chlamydomonas cytochrome f (cyt f) with either Chlamydomonas plastocyanin (PC) or Chlamydomonas cytochrome c(6) (cyt c(6)) was studied using Brownian dynamics simulations. The two electron acceptors (PC and cyt c(6)) were found to be essentially interchangeable despite a lack of sequence homology and different secondary structures (beta-sheet for PC and alpha-helix for cyt c(6)). Simulations using PC and cyt c(6) interacting with cyt f showed approximately equal numbers of successful complexes and calculated rates of electron transfer. Cyt f-PC and cyt f-cyt c(6) showed the same types of interactions. Hydrophobic residues surrounding the Y1 ligand to the heme on cyt f interacted with hydrophobic residues on PC (surrounding the H87 ligand to the Cu) or cyt c(6) (surrounding the heme). Both types of complexes were stabilized by electrostatic interactions between K65, K188, and K189 on cyt f and conserved anionic residues on PC (E43, D44, D53, and E85) or cyt c(6) (E2, E70, and E71). Mutations on cyt f had identical effects on its interaction with either PC or cyt c(6). K65A, K188A, and K189A showed the largest effects whereas residues such as K217A, R88A, and K110A, which are located far from the positive patch on cyt f, showed very little inhibition. The effect of mutations observed in Brownian dynamics simulations paralleled those observed in experiments.     

Reversibility of structural transition of cytochrome c on interacting with and releasing from alternating copolymers of maleic Acid and alkene

The interaction of cytochrome c (cyt c) with poly(isobutylene-alt-maleic acid) (PIMA) and poly(1-tetradecene-alt-maleic acid) (PTMA) was studied using circular dichroism, absorption spectroscopy, and atomic force microscopy to investigate the electrostatic and hydrophobic influence of the copolymers on the structure of cyt c. At pH 7.4, the interaction of PIMA with cyt c can only partly disturb the integrity of the heme pocket, while PTMA has very intensive influence on the structure of cyt c. After adding 0.15 M NaCl, PIMA-cyt c complexes dissociate, and the released cyt c recovers its native structure, whereas NaCl has no significant influence on PTMA-cyt c complexes. GuHCl (0.5 M) destroys PTMA-cyt c complexes, forming GuHCl-PTMA precipitates; the cyt c released from the complexes regenerates its native structure. In comparison with electrostatic interaction, hydrophobic interaction leads to more stable polymer-cyt c complexes and more intensive influence on cyt c structure, but cyt c can recover its native state after release.     

Double mutant studies identify electrostatic interactions that are important for docking cytochrome c2 onto the bacterial reaction center

Cytochrome c2 (cyt) is the mobile electron donor to the reaction center (RC) in photosynthetic bacteria. The electrostatic interactions involved in the dynamics of docking of cyt onto the RC were examined by double mutant studies of the rates of electron transfer between six modified Rhodobacter sphaeroides RCs in which negatively charged acid residues were replaced with Lys and five modified Rhodobacter capsulatus Cyt c2 molecules in which positively charged Lys residues were replaced with Glu. We measured the second-order rate constant, k2, for electron transfer from the reduced cyt to the oxidized primary donor on the RC, which reflects the energy of the transition state for the formation of the active electron transfer complex. Strong interactions were found between Lys C99 and Asp M184/Glu M95, and between Lys C54 and Asp L261/Asp L257. The interacting residues were found to be located close to each other in the recently determined crystal structure of the cyt-RC complex [Axelrod, H., et al. (2002) J. Mol. Biol. (in press)]. The interaction energies were approximately inversely proportional to the distances between charges. These results support earlier suggestions [Tetreault, M., et al. (2001) Biochemistry 40, 8452-8462] that the structure of the transition state in solution resembles the structure of the cyt-RC complex in the cocrystal and indicate that specific electrostatic interactions facilitate docking of the cyt onto the RC in a configuration optimized for both binding and electron transfer. The specific interaction between Asp M184 and Lys C99 may help to nucleate short-range hydrophobic contacts.     

The alpha-helical membrane spanning domain of cytochrome b5 interacts with cytochrome P450 via nonspecific interactions

Cytochrome b5 (cyt b5) is an amphipathic membrane-bound heme protein found in the endoplasmic reticulum of eukaryotes. It consists of three domains, an N-terminal cytosolic, hydrophilic domain containing the heme, a short flexible linker and an alpha-helical membrane-spanning domain. This study investigated whether there are specific side chain helix-helix packing interactions between the COOH-terminal membrane anchor of cyt b5 and cytochrome P450 (cyt P450) 2B4 in a purified reconstituted system. Alanine was inserted at six positions in the membrane anchor of cyt b5. Insertion of alanine into an alpha-helix causes all amino acids at its carboxyl terminus to be rotated by 100 degrees . The ability of the alanine insertion mutants of cyt b5 to bind to cyt P450 2B4 was similar to that of the wild-type protein as was the ability of the mutant cyts b5 to stimulate the metabolism of the anesthetic, methoxyflurane. These results demonstrate that the C-terminal hydrophobic alpha-helix of cyt b5 does not interact with cyt P450 2B4 through a specific stereochemical fit of amino acid side chains, but rather through nonspecific interactions.     

Hydrophobic effect of trityrosine on heme ligand exchange during folding of cytochrome c

Effect of a hydrophobic peptide on folding of oxidized cytochrome c (cyt c) is studied with trityrosine. Folding of cyt c was initiated by pH jump from 2.3 (acid-unfolded) to 4.2 (folded). The Soret band of the 2-ms transient absorption spectrum during folding decreased its intensity and red-shifted from 397 to 400 nm by interaction with trityrosine, whereas tyrosinol caused no significant effect. The change in the transient absorption spectrum by interaction with trityrosine was similar to that obtained with 100 mM imidazole, which showed that the population of the intermediate His/His coordinated species increased during folding of cyt c by interaction with trityrosine. The absorption change was biphasic, the fast phase (82+/-9s(-1)) corresponding to the transition from the His/H(2)O to the His/Met coordinated species, whereas the slow phase (24+/-3s(-1)) from His/His to His/Met. By addition of trityrosine, the relative ratio of the slow phase increased, due to increase of the His/His species at the initial stage of folding. According to the resonance Raman spectra of cyt c, the high-spin 6-coordinate and low-spin 6-coordinate species were dominated at pH 2.3 and 4.2, respectively, and these species were not affected by addition of trityrosine. These results demonstrated that the His/His species increased by interaction with trityrosine at the initial stage of cyt c folding, whereas the heme coordination structure was not affected by trityrosine when the protein was completely unfolded or folded. Hydrophobic peptides thus may be useful to study the effects of hydrophobic interactions on protein folding.     

Stable submolecular folding units in a non-compact form of cytochrome c.

Studies of structure, dynamics, and stability of cytochrome c (cyt c) at low pH in a non-compact pre-molten globule state indicate that the protein contains submolecular folding units that are independently stable. In high salt, acid cyt c (pD 2.2; where D is deuterium) is nearly as compact as the native form. Nuclear magnetic resonance (n.m.r.) line broadening typical of the molten globule form is seen, indicating loosened packing and increased mobility not only for side-chains but also for the main chain. As NaCl concentration is decreased below 0.05 M, cyt c expands due to the deshielding of electrostatic repulsions, attaining a linear extent perhaps double that of the native protein (viscosity, fluorescence). In the extended form, tertiary structural hydrogen bonds are largely broken (hydrogen exchange rate), some normally buried parts of the protein are exposed to water (fluorescence), and many of the native side-chain contacts must be lost. Nevertheless, almost all of the helical content is retained (circular dichroism). The helices involve the same amino acid residues that are helical in the native state (hydrogen exchange labeling monitored by 2-dimensional n.m.r.). The equilibrium constant for helix formation at 20 degrees C (0.02 M-NaCl, pD 2.2) is about 10 (hydrogen exchange rate), even though the individual helical segments when isolated have little or no structure. Additional experiments were done to check assumptions and calibrate parameters that underlie the hydrogen exchange analysis of protein folding. These results indicate that the native-like helical segments in the expanded non-globular form of cyt c exist as part of somewhat larger submolecular folding units that possess significant equilibrium stability. Results from equilibrium and kinetic studies of protein folding support the generality of this conclusion. This view is contrary to the two-state paradigm for equilibrium folding and inconsistent with the idea that side-chain packing constraints determine folding motifs. The result suggests an extension of the thermodynamic hypothesis for protein structure to kinetic folding processes, so that the amino acid code for equilibrium and kinetic folding may be the same, and also seems pertinent to the biological evolution of contemporary protein structures.     

Cytochrome c impaled: investigation of the extended lipid anchorage of a soluble protein to mitochondrial membrane models

Cyt c (cytochrome c) has been traditionally envisioned as rapidly diffusing in two dimensions at the surface of the mitochondrial inner membrane when not engaged in redox reactions with physiological partners. However, the discovery of the extended lipid anchorage (insertion of an acyl chain of a bilayer phospholipid into the protein interior) suggests that this may not be exclusively the case. The physical and structural factors underlying the conformational changes that occur upon interaction of ferrous cyt c with phospholipid membrane models have been investigated by monitoring the extent of the spin state change that result from this interaction. Once transiently linked by electrostatic forces between basic side chains and phosphate groups, the acyl chain entry may occur between two parallel hydrophobic polypeptide stretches that are surrounded by positively charged residues. Alteration of these charges, as in the case of non-trimethylated (TML72K) yeast cyt c and Arg91Nle horse cyt c (where Nle is norleucine), led to a decline in the binding affinity for the phospholipid liposomes. The electrostatic association was sensitive to ionic strength, polyanions and pH, whereas the hydrophobic interactions were enhanced by conformational changes that contributed to the loosening of the tertiary structure of cyt c. In addition to proposing a mechanistic model for the extended lipid anchorage of cyt c, we consider what, if any, might be the physiological relevance of the phenomenon.     

Investigations on the insertion of the mitochondrial precursor protein apocytochrome c into model membranes

Different aspects of the interaction of apocytochrome c and model membranes composed of negatively charged lipids, were studied in order to get insight into the nature of this interaction. The effect of the protein on the lipid packing properties are revealed by DSC, ESR and monolayer techniques. These experiments clearly demonstrate that upon electrostatic interaction with the negatively charged phospholipids, apocytochrome c is able to penetrate into the hydrophobic region of the model membrane. In the case of 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, this results in a perturbation of 160 lipid molecules per apocytochrome c molecule. Most likely, apocytochrome c disrupts the formation of the gel phase and restricts the lipid chain motion above the gel to liquid-crystalline phase transition. Tryptophan fluorescence measurements confirm that at least a part of the protein penetrates into the bilayer, and suggest that after this penetration, the tryptophan (residue no. 59) is located in the glycerol backbone region of the phospholipids. Although the secondary structure of apocytochrome c is predicted to contain about 35% of alpha-helical structure, the CD pattern of an aqueous solution of the protein is featureless. However, negatively charged lipids are able to express this alpha-helical potency in the apocytochrome c, which might be important for the insertion of the protein into lipid membranes.     

Structural analysis of zinc-substituted cytochrome c

Zinc-substituted cytochrome c has been widely used in studies of protein-protein interactions and photo-induced electron transfer reactions between proteins. However, the coordination geometry of zinc in zinc-substituted cyt c has not yet been determined; two different opinions about the coordination have been reached. Here the solution structures of zinc-substituted cytochrome c that might be five-coordinated and six-coordinated have been refined separately by using (1)H NMR spectroscopy, and the zinc coordination geometry was determined just by NOE distance constraints. Structural analysis of the energy-minimized average solution structures of both the pentacoordinated and hexacoordinated geometries indicate that that zinc in zinc-substituted cyt c should be bound to both His18 and Met80, which means that the zinc is six-coordinated. RMSD values of the family of 25 six-coordinated structures from the average structure are 0.66+/-0.13 A and 1.09+/-0.16 A for the backbone and all heavy atoms, respectively. A statistical analysis of the structure indicates its satisfactory quality. Comparison of the solution structure of the six-coordinated energy-minimized average structure of zinc-substituted cytochrome c with the solution structure of reduced cytochrome c reveals that for the overall folding the secondary structure elements are very close. The availability of the structure provides for a better understanding of the protein-protein complex and for electron transfer processes between Zn cyt c and other metalloproteins.     

Ultraviolet Raman examination of the environmental dependence of bombolitin I and bombolitin III secondary structure.

Bombolitin I and III (BI and BIII) are small amphiphilic peptides isolated from bumblebee venom. Although they exist in predominately nonhelical conformations in dilute aqueous solutions, we demonstrate, using UV Raman spectroscopy, that they become predominately alpha-helical in solution at pH > 10, in high ionic strength solutions, and in the presence of trifluoroethanol (TFE) and dodecylphosphocholine (DPC) micelles. In this paper, we examine the effects of electrostatic and hydrophobic interactions that control folding of BI and BIII by systematically monitoring their secondary structures as a function of solution conditions. We determine the BI and BIII secondary structure contents by using the quantitative UV Raman methodology of Chi et al. (1998. Biochemistry. 37:2854-2864). Our findings suggest that the alpha-helix turn in BIII at neutral pH is stabilized by a salt bridge between residues Asp2 and Lys5. This initial alpha-helical turn results in different BI and BIII alpha-helical folding mechanisms observed in high pH and high salt concentrations: BIII folds from its single alpha-helix turn close to its N-terminal, whereas the BI alpha-helix probably nucleates within the C-terminal half. We also used quasielastic light scattering to demonstrate that the BI and BIII alpha-helix formation in 0.2 M Ca(ClO4)2 is accompanied by formation of trimers and hexamers, respectively.     

Effects of charged amino-acid mutation on the solution structure of cytochrome b5 and binding between cytochrome b5 and cytochrome c

The solution structure of oxidized bovine microsomal cytochrome b(5) mutant (E48, E56/A, D60/A) has been determined through 1524 meaningful nuclear Overhauser effect constraints together with 190 pseudocontact shift constraints. The final family of 35 conformers has rmsd values with respect to the mean structure of 0.045+/-0.009 nm and 0.088+/-0.011 nm for backbone and heavy atoms, respectively. A characteristic of this mutant is that of having no significant changes in the whole folding and secondary structure compared with the X-ray and solution structures of wild-type cytochrome b(5). The binding of different surface mutants of cytochrome b(5) with cytochrome c shows that electrostatic interactions play an important role in maintaining the stability and specificity of the protein complex formed. The differences in association constants demonstrate the electrostatic contributions of cytochrome b(5) surface negatively charged residues, which were suggested to be involved in complex formation in the Northrup and Salemme models, have cumulative effect on the stability of cyt c-cyt b(5) complex, and the contribution of Glu48 is a little higher than that of Glu44. Moreover, our result suggests that the docking geometry proposed by Northrup, which is involved in the participation of Glu48, Glu56, Asp60, and heme propionate of cytochrome b(5), do occur in the association between cytochrome b(5) and cytochrome c.     

Protein interactions leading to conformational changes monitored by limited proteolysis: apo form and fragments of horse cytochrome c

Proteolysis experiments have been used to monitor the conformational transitions from an unfolded to a folded state occurring when the apo form of horse cytochrome c (cyt c) binds the heme moiety or when two fragments of cyt c form a native-like 1:1 complex. Proteinase K was used as a proteolytic probe, in view of the fact that the broad substrate specificity of this protease allows digestion at many sites along a polypeptide chain. The rather unfolded apo form of cyt c binds heme with a concomitant conformational transition to a folded species characterized by an enhanced content of helical secondary structure. While the holoprotein is fully resistant to proteolytic digestion and the apoprotein is digested to small peptides, the noncovalent complex of the apoprotein and heme exhibits an intermediate resistance to proteolysis, in agreement with the fact that the more folded structure of the complex makes the protein substrate more resistant to proteolysis. The noncovalent native-like complex of the two fragments 1-56 and 57-104 of cyt c, covering the entire polypeptide chain of 104 residues of the protein, is rather resistant to proteolysis, while the individual fragments are easily digested. Fragment 57-104 is fast degraded to several peptides, while fragment 1-56 is slowly degraded stepwise from its C-terminal end, leading initially mostly to fragments 1-48 and 1-40 and, at later stages of proteolysis, fragments 1-38, 1-35, 1-33, and 1-31. Thus, proteolysis data indicate that the heme containing fragment 1-56 has a rather compact core and a C-terminal flexible tail. Upon prolonged incubation of the complex of fragments 1-56 and 57-104 (nicked cyt c) with proteinase K, a chain segment is removed from the nicked protein, leading to a gapped protein complex of fragments of 1-48 and 57-104 and, on further digestion, fragments 1-40 and 57-104. Of interest, the chain segment being removed by proteolysis of the complex matches the omega-loop which is evolutionarily removed in cyt c of microbial origin. Overall, rates and/or resistance to proteolysis correlates well with the extent of folding of the protein substrates, as deduced from circular dichroism measurements. Thus, our results underscore the utility of proteolytic probes for analyzing conformational and dynamic features of proteins. Finally, a specific interest of the cyt c fragment system herewith investigated resides in the fact that the fragments are exactly the exon products of the cyt c gene.