Carmichael J. A. Wallace

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.

Peptide Ligation and Semisynthesis

Semisynthesis is used to create defined analogues of proteins by the chemical manipulation of peptide fragments largely derived from the natural protein and the subsequent reassembly of those fragments into a near-native conformation. In common with the total synthesis of proteins, it requires efficient and non-destructive methods for peptide religation. Recently, a wide range of chemoselective ligation schemes have been elaborated that now permit the assembly of minimally protected peptides from either synthetic or natural sources.

Synergy in Protein Engineering MUTAGENIC MANIPULATION OF PROTEIN STRUCTURE TO SIMPLIFY SEMISYNTHESIS

Semisynthesis is a chemical technique of protein engineering that provides a valuable complement to directed mutagenesis. It is the method of choice when the structural modification requires, for example, a noncoded amino acid. The process involves specific and limited protein fragmentation, structural manipulation of the target sequence, and subsequent religation of fragments to give the mutant holoprotein. We suggested and demonstrated that mutagenesis and semisynthesis could be used synergistically to achieve protein engineering goals otherwise unobtainable, if mutagenesis was used to shuffle methionine residues in the yeast cytochrome c sequence (Wallace, C. J. A., Guillemette, J. G., Hibiya, Y., and Smith, M. (1991) J. Biol. Chem. 266, 21355-21357). These residues can not only be sites of specific cleavage by CNBr but also of spontaneous peptide bond synthesis between fragments in noncovalent complexes, which greatly facilitates the semisynthetic process. We have now used an informed “methionine scan” of the protein sequence to discover other useful sites and to characterize the factors that promote this extraordinary and convenient autocatalytic religation. Of eight sites canvassed, in a wide range of settings, five efficiently provoked peptide bond synthesis. The principal factor determining efficiency seems to be the hydropathy of the religation site. The mutants created have also provided some new insights on structure-function relationships in the cytochrome.

Resolving the Individual Components of a pH-Induced Conformational Change

This communication introduces a simple method to determine the pKs of microscopic ionizations from complex titration curves. We used this approach to study the alkaline transition (pH-dependent ligand exchange) of mitochondrial cytochrome c. The linearization of titration curves permitted resolution of two to three limiting microscopic ionizations. By combining these data with studies of the temperature dependence of ligand-exchange equilibria, we found evidence that the alkaline transition comprises two chemically distinct processes: the deprotonation of the alternative ligands and the break of the iron-methionine ligation bond. We also noted that, in the horse and untrimethylated S. cerevisiae iso-1 cytochromes c, the permissible deprotonation of the epsilon-amino group of Lys(72) allows formation of an alkaline isomer at lower pH, with lesser stability, which leads to hysteresis in the titration curves. The linearization of the titration curves for different cytochromes c thus brings insight on the microscopic contributions to conformational stability.

Definition of a Nucleotide Binding Site on Cytochrome c by Photoaffinity Labeling

We have used TNP-8N3-AMP (2′(3′)-O-(2,4,6-trinitrophenyl)-8-azidoadenosine monophosphate) and TNP-8N3-ATP to probe the ATP binding site(s) of cytochrome c. Irradiation of cytochrome c with close to stoichiometric amounts of TNP-8N3-AMP at low ionic strength derivatized approximately half of the protein, with the mono-derivatized species being associated with four peaks (B, 6%; C, 17%; D, 24%; E, 4%) eluted from a cation exchange column. Irradiation in the presence of ATP suggested that the main peaks C and D resulted from more specific nucleotide binding. Thermolysin digestion and TNP-peptide purification and sequencing revealed that peak C was associated with derivatization of mainly Lys-86 and to a lesser extent Lys-72 and peak D with mainly Lys-87 and less so with Lys-72. Minor peaks B and E could not be identified. TNP-8N3-ATP photolabeling produced similar results, showing favored interaction of the adenyl ring with Lys-86 and Lys-87 and to a lesser extent with Lys-72. The results are compatible with previous findings that suggest that the principal locus of ATP binding is at nearby Arg-91 (Corthesy, B. E., and Wallace, C. J. A. (1986) Biochem. J. 236, 359-364). Molecular modeling with energy-minimized docking of ATP between the 60s helix and the 80s stretch with the γ-phosphate constrained to interact with Arg-91, places the 8 position close to Lys-86 and Lys-87 in the anti conformation about the glycosidic bond and to Lys-72 in the syn conformation, and the ribose hydroxyls within H-bonding distance of Glu-69.

Probing the role of the conserved beta-II turn Pro-76/Gly-77 of mitochondrial cytochrome c.

The loop segment comprising residues 70-84 in mitochondrial cytochrome c serves to direct the polypeptide backbone to permit the functionally required heme Fe - S (Met-80) co-ordination. The primary sequence here is highly conserved, which is something rarely observed in surface loop segments and suggests that its purpose is more complex than its obvious structural role. The beta-II turn formed by Pro-76 and Gly-77 is postulated to be key to the redirection of the peptide backbone required to execute the loop. We assessed the importance of Pro-76 and Gly-77 by mutating 1 or both of these residues to alanine such that the range of allowable dihedral angles was altered, and this resulted in significant changes in physicochemical properties and biological activities. We observed structural perturbations using circular dichroism spectroscopy and thermal denaturation studies. Based on these changes, we propose that the Pro-76/Gly-77 beta-II turn precisely orients the 70s loop, not only to maintain the backbone orientation required for the formation of the axial heme ligand, but also to provide a complementary surface to physiological partners.

Synergy of Semisynthesis and Site-Directed Mutagenesis: Manipulating Genes to Create Sites for Autocatalytic Protein Fragment Religation

Publisher Summary Semisynthesis is a means to protein engineering that relies, as the name implies, on the use of a large proportion of the natural protein in the assembly of the synthetic product. In a typical fragment condensation semisynthesis, the protein under study is side-chain protected and cleaved, chemically or enzymatically, into a limited number of fragments that will then be isolated and purified. The fragment that contains the residue(s) of interest will be modified to introduce the desired substitution. This may involve total chemical synthesis of the fragment, its replacement by a corresponding peptide from another species, sequential degradation and resynthesis of the natural fragment, or fragment-specific chemical modification. Other peptides may be further protected or modified to facilitate the next step, fragment condensation. Product isolation will be followed by deprotection and purification. The two main practical problems associated with such schemes are a requirement for relatively large amounts of protein and the difficulty of the fragment condensation step. This chapter discusses combination of semisynthesis and directed mutagenesis to create idealized sites.

Using semisynthesis to insert heavy-atom labels in functional proteins

Publisher Summary This chapter describes the method to incorporate residues, including heavy atoms (such as selenium and bromine), into cytochrome c for experiments using X-ray standing waves to visualize protein orientation. In protein semisynthesis, fragments of the natural protein are used as preformed intermediates in the synthesis of a mutant structure. Semisynthesis can be used to introduce d-amino acids, labels for spectroscopy at specific sites, or other non-coded side chain structures. While heavy-atom derivatives are useful in conventional X-ray crystallographic determinations of protein three-dimensional structure, this new technique is absolutely dependent on the presence of an atom that is X-ray fluorescent at the frequencies generated in synchrotron radiation. With two heavy atoms, two distances can be measured and a precise orientation determined. Because artificial membranes can be layered on the mirror surface, the method is ideally suited for the studies of the interaction of membranes and associated proteins or peptide hormones.