The localization of pigment-binding polypeptides in membranes of Rhodopseudomonas viridis

Abstract Inside-out and right-side out vesicles were isolated from the intracytoplasmic membrane system of the photosynthetic bacterium Rhodopseudomonas viridis and treated with proteinase K. Afterwards the pigment-binding proteins of the photosynthetic apparatus were extracted from the membrane, purified and the N- and C-terminal amino acyl sequences determined.
Forty-eight amino acids were found to be removed from the N-terminal domain of the M-subunit and twenty-eight amino acids split off the L-subunit of reaction center when inside-out vesicles were digested with proteinase K.
Six amino acids of the N-terminal region of the beta polypeptide of the light-harvesting complex B1020 were removed when inside-out vesicles were treated with proteinase K. The N-terminal domains of alpha and gamma polypeptides of the antenna complex B1020 were not cleaved by proteinase K either in right-side out or in inside-out vesicles. It is concluded that the N-terminal domains of M-, L- and β-subunits are exposed and accessible to proteinase K on the cytoplasmic surface of the membrane. This is in agreement with results obtained with other photosynthetic bacteria. The orientation of the other light-harvesting polypeptides is discussed.     

Localization of reaction center and B800-850 antenna pigment proteins in membranes of Rhodobacter sphaeroides.

The localization of the N- and C-terminal regions of pigment-binding polypeptides of the bacterial photosynthetic apparatus of Rhodobacter sphaeroides was investigated by proteinase K treatment of chromatophore and spheroplast-derived vesicles and amino acid sequence determination. Under conditions of proteinase K treatment of chromatophores, which left the in vivo absorption spectrum and the membrane intact, 15 and 46 amino acyl residues from the N-terminal regions of the L and M subunits, respectively, of the reaction center polypeptides were removed. The N termini are therefore exposed on the cytoplasmic surface of the membrane. The C-terminal domain of the light-harvesting B800-850 alpha and B870 alpha polypeptides was found to be exposed on the periplasmic surface of the membrane. A total of 9 and 13 amino acyl residues were cleaved from the B800-850 alpha and B870 alpha polypeptides, respectively, when spheroplasts were treated with proteinase K. The N-terminal regions of the alpha polypeptides were not digested in either membrane preparation and were apparently protected from proteolytic attack. Seven N-terminal amino acyl residues of the B800-850 beta polypeptide were removed after the digestion of chromatophores. C-terminal residues were not removed after the digestion of chromatophores or spheroplasts. The C termini seem to be protected from protease attack by interaction with the membrane. Therefore, the N-terminal regions of the beta polypeptides are exposed on the cytoplasmic membrane surface. The C termini of the beta polypeptides are believed to point to the periplasmic space.     

Probing the topology of proteins in the chromatophore membrane of Rhodospirillum rubrum G-9 with proteinase K

All the major membrane proteins of isolated chromatophore vesicles are eventually degraded upon incubation with the unspecific proteinase K. These proteins must therefore be exposed at least partially or temporarily on the cytosolic surface of the membrane which is exclusively accessible to the proteinase in intact chromatophore vesicles. That the vesicles are intact during the incubation with proteinase is demonstrated by the finding that cytochrome c₂, which is located in the interior of the vesicles, is protected from proteolytic attack. The degree of degradation of the various chromatophore proteins and the time taken for degradation differ characteristically. From the changes in intensity of the gel bands during the course of digestion it appears that reaction center subunit H is digested first, much faster than are subunits M and L. The near-infrared absorption spectrum of the chromatophores changes only after proteolytic degradation of these two pigment-carrying subunits. Fading of the band of the light-harvesting polypeptide is evident only after prolonged incubation. It seems that this is the most stable component of the chromatophore membrane. The light-harvesting polypeptide appears to be somewhat shortened eventually, leaving the protein conformation necessary for holding the pigments unchanged, as shown by the absorption spectrum. The possible topology of these major membrane components is discussed in the light of these findings.     

The light-harvesting polypeptides of Rhodospirillum rubrum. II. Localisation of the amino-terminal regions of the light-harvesting polypeptides B 870-alpha and B 870-beta and the reaction-centre subunit L at the cytoplasmic side of the photosynthetic membrane of Rhodospirillum rubrum G-9+

The unspecific proteinase K and the specific proteases alpha-chymotrypsin, trypsin and S. aureus V 8 protease were used in order to determine the orientation of the polypeptides B 870-alpha and B 870-beta from the major antenna complex B 870 of Rs. rubrum G-9+ within the chromatophore membrane (inside-out vesicle). Although B 870-alpha exhibits cleavable peptide bonds, treatment with specific proteases yielded splitting only in B 870-beta within the N-terminal region. In the case of proteinase K, which was most effective, mainly 6 (B 870-alpha) and 16 (B 870-beta) amino acid residues were removed from their N-terminal parts as proved by means of Edman degradation of cleavage products. The major peptide bonds cleaved were identified as Gln6-Leu7 in B 870-alpha and as Lys16-Glu17 in B 870-beta. The central hydrophobic stretch regions and the relatively hydrophilic C-terminal parts of both light-harvesting polypeptides were not affected by proteinase K. On the basis of these degradation experiments a transmembrane orientation of B 870-alpha and B 870-beta is postulated, with their N-terminal towards the cytoplasm and their C-termini towards periplasm with regard to the photosynthetic membrane. This hypothesis is supported by the transmembrane model proposed by Brunisholz et al. (Hoppe-Seyler's Z., Physiol. Chem., (1984) 365, 675-688) in which the hydrophobic stretch of B 870-alpha and of B 870-beta forming an alpha-helix would span the membrane once. Organic solvent extraction of chromatophores treated with proteinase K yielded a fairly pure polypeptide fragment with an apparent molecular mass of 14000 Da. Its N-terminal amino-acid sequence is identical with the sequence within the N-terminal region of the reaction centre subunit L of Rs. rubrum G-9+. Thus it is most likely that as in the case of B 870-beta, proteinase K removed 16 amino acid residues from the N-terminal part of subunit L. This subunit therefore also seems to be exposed at the surface of the cytoplasmic side of the chromatophore membrane.     

ATP-synthetase complex (F1F0) from Escherichia coli. Purification and characterization of subunits A and B of the F0 part

An intact B890 light-harvesting pigment—protein complex has been obtained from Rhodospirillum rubrum strain S1. We show that this complex contains two types of low-M_r polypeptide. Both these polypeptides are present in the intact chromatophore membrane. Analysis of the pigment content of this complex suggests that per pair of polypeptides the complex contains 2 molecules of bacteriochlorophyll and one molecule of carotenoid.     

Transport of α-aminoisobutyrate by cells and membrane vesicles of Pseudomonas fluorescens

The transport of α-aminoisobutyrate into Pseudomonas fluorescens NCIB 8865 and membrane vesicles prepared from this organism has been studied. Uptake by cells was mediated by two active transport systems with different apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values, while transport into membrane vesicles was mediated by a single component. The effect of inhibitors on the energy-coupling mechanism for α-aminoisobutyrate transport in these systems suggests that a membrane potential may play a significant role in supporting α-aminoisobutyrate transport. The magnitude of the membrane potential in the vesicle system, and the sensitivity of its generation to inhibitors, has been measured using ¹³⁷Cs in the presence of valinomycin. Direct attempts to demonstrate a proton-symport mechanism for α-aminoisobutyrate transport were negative.     

Na+/solute symport in membrane vesicles from Bacillus alcalophilus

The characteristics of α-aminoisobutyric acid translocation were examined in membrane vesicles from obligately alkalophilic Bacillus alcalophilus and its non-alkalophilic mutant derivative, KM23. Vesicles from both strains exhibited α-aminoisobutyric acid uptake upon energization with ascorbate and N,N,N′,N′-tetramethyl-p-phenylenediamine. The presence of Na⁺ caused a pronounced reduction in the Km for α-aminoisobutyric acid in wild-type but not KM23 vesicles; the maximum velocity (V) was unaffected in vesicles from both strains. Passive efflux and exchange of α-aminoisobutyric acid from wild-type vesicles were Na⁺-dependent and occurred at comparable rates (with efflux slightly faster than exchange). This latter observation suggests that the return of the unloaded carrier to the inner surface is not rate-limiting for efflux. The rates of α-aminoisobutyric acid efflux and exchange were also comparable in KM23 vesicles, but were Na⁺-independent. Furthermore, in vesicles from the two strains, both efflux and exchange were inhibited by generation of a transmembrane electrochemical gradient of protons, outside positive. This suggests that the ternary complex between solute, carrier, and coupling ion bears a positive charge in both strains even though the coupling ion is changed. Evidence from experiments with an alkalophilic strain that was deficient in l-methionine transport indicated that the porters, i.e., the solute-translocating elements, used by non-alkalophilic mutants are not genetically distinct from those used by the alkalophilic parent; that is, the change in coupling ion cannot be explained by the expression of a completely new set of Na⁺-independent, H⁺-coupled porters upon mutation of B. alcalophilus to non-alkalophily.     

Sidedness of membrane structures in Rhodopseudomonas sphaeroides. Electrochemical titration of the spectrum changes of carotenoid in spheroplasts,spheroplast membrane vesicles and chromatophores

The shift of the carotenoid absorption spectrum induced by illumination and valinomycin-K⁺ addition was investigated in membrane structures with different characteristics and opposite sidednesses isolated from Rhodopseudomonas sphaeroides. Right-side-out membrane structures were prepared by isotonic lysozyme-EDTA treatment of the cells (spheroplasts) and by hypotonic treatment of spheroplasts (spheroplast membrane vesicles). Inside-out membrane structures (“chromatophores”) were obtained by treating spheroplast membrane vesicles by French press or sonication.The membrane structures with either sidedness showed the same light-induced change of the “red shift” type. However, the absorbance change by K⁺ addition in the presence of valinomycin in the right-side-out membrane structures were opposite to that in the inverted vesicles, “blue shift” in the former and “red shift” in the latter. The carotenoid absorbance change was linear to membrane potential, calculated from the concentration of KCl added, with a reference on the cytoplasmic side, through positive and negative ranges.     

Orientation of the cytoplasmically made subunits of beef heart cytochrome c oxidase determined by protease digestion and antibody binding experiments

The topology of several of the cytoplasmically made subunits of beef heart cytochrome c oxidase has been determined by protease digestion of oriented membrane preparations, using subunit-specific antibodies to identify cleavage products. Reconstituted vesicles of cytochrome c oxidase and asolectin were used as a vesicle preparation with the C domain of the enzyme available for protease digestion. Submitochondrial particles were used as vesicles with the M domain outermost. Trypsin and/or proteinase K cleaved polypeptides CIV, ASA, AED, STA, and IHQ. Cleavage of CIV, STA, and IHQ was from the M domains only and involved the removal of a fragment from the N-terminus in each case. Polypeptide AED was cleaved from the C side in the N-terminal part, while ASA was cleaved from both the C and M domains. Polypeptide fragments were electroblotted from polyacrylamide gels onto derivatized glass paper and sites of proteolytic cleavage determined by N-terminal sequence analysis.     

The core of Ure2p prion fibrils is formed by the N-terminal segment in a parallel cross-β structure: evidence from solid-state NMR

Intracellular fibril formation by Ure2p produces the non-Mendelian genetic element [URE3] in Saccharomyces cerevisiae, making Ure2p a prion protein. We show that solid-state NMR spectra of full-length Ure2p fibrils, seeded with infectious prions from a specific [URE3] strain and labeled with uniformly ¹⁵N-¹³C-enriched Ile, include strong, sharp signals from Ile residues in the globular C-terminal domain (CTD) with both helical and nonhelical ¹³C chemical shifts. Treatment with proteinase K eliminates these CTD signals, leaving only nonhelical signals from the Gln-rich and Asn-rich N-terminal segment, which are also observed in the solid-state NMR spectra of Ile-labeled fibrils formed by residues 1-89 of Ure2p. Thus, the N-terminal segment, or “prion domain” (PD), forms the fibril core, while CTD units are located outside the core. We additionally show that, after proteinase K treatment, Ile-labeled Ure2p fibrils formed without prion seeding exhibit a broader set of solid-state NMR signals than do prion-seeded fibrils, consistent with the idea that structural variations within the PD core account for prion strains. Measurements of ¹³C-¹³C magnetic dipole-dipole couplings among ¹³C-labeled Ile carbonyl sites in full-length Ure2p fibrils support an in-register parallel β-sheet structure for the PD core of Ure2p fibrils. Finally, we show that a model in which CTD units are attached rigidly to the parallel β-sheet core is consistent with steric constraints.     

Topology of diphtheria toxin in lipid vesicle membranes: a proteolysis study

The diphtheria toxin (DT) membrane topology was investigated by proteolysis experiments. Diphtheria toxin was incubated with asolectin liposomes at pH 5 in order to promote its membrane insertion, and the protein domains located outside the lipid vesicles were digested with proteinase K (which is a non-specific protease). The protected peptides were separated by electrophoresis and identified by microsequence analysis. Their orientation with respect to the lipid bilayer and their accessibility to the aqueous phase were determined by attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR). These data, combined with those provided by proteolytic cleavage with a specific protease (endoproteinase Glu-C), led us to propose a topological model of the N-terminal part of the diphtheria toxin B fragment inserted into the lipid membrane. In this model, two a-helices adopt a transmembrane orientation, with their axes parallel to the lipid acyl chains, while a third o-helix could adopt a transmembrane topology only in a small proportion of DT molecules.     

Study of the irreversible binding of Bacillus thuringiensis Cry1Aa to brush border membrane vesicles from Bombyx mori midgut

The binding of Bacillus thuringiensis δ-endotoxin to brush border membrane vesicles (BBMVs) from the target insect larval midgut comprises with not only a reversible but also an irreversible component. The irreversible binding of δ-endotoxin is thought to be a pathologically important factor. Here, we studied the irreversible binding of Cry1Aa to the BBMVs of Bombyx mori. The ¹²⁵I-labeled Cry1Aa bound to the solubilized brush border membrane (BBM) through rapid dissociation only, unlike the binding to BBMVs, indicating that the toxin bound to the solubilized BBM through only a reversible process. Low-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the toxin bound irreversibly to BBMVs formed an oligomer of 220 kDa, whereas that bound reversibly to the solubilized BBM did not oligomeraize. When the ¹²⁵I-labeled Cry1Aa bound irreversibly to the BBMVs was digested by proteinase K, approximately 40% of the toxin observed to be resistant to proteinase K. The molecular mass of the toxin resistant to proteinase K was 60 kDa, suggesting that the irreversible binding comprise two forms. These results support the notion that the irreversible binding of the toxin to BBMVs is due to the insertion of the toxin into the lipid bilayers and oligomerization to form channels.     

Pigment organization of the B800–850 antenna complex of Rhodopseudomonas sphaeroides

The B800–850 antenna complex of Rhodopseudomonas sphaeroides was studied by comparing the spectral properties of several different types of complexes, isolated from chromatophores by means of the detergents lithium dodecyl sulfate (LDS) or lauryl dimethylamine N-oxide (LDAO). Fluorescence polarization spectra of the BChl 800 emission at 4 K indicated that rapid energy transfer between at least two BChl 800 molecules occurs with a rate constant of energy transfer k_ET > 3 · 10¹² s^?1. The maximal dipole-dipole distance between the two BChl 800 molecules was calculated to be 18–19 Å. The porphyrin rings of the BChl 800 molecules are oriented parallel to each other, while their Q_y transition moments are mutually perpendicular. The energy-transfer efficiency from carotenoid to bacteriochlorophyll measured in different complexes showed that two functionally different carotenoids are present associated with, respectively, BChl 800 and BChl 850. Fluorescence polarization and linear dichroism spectra revealed that these carotenoids have different absorption spectra and a different orientation with respect to the membrane. The carotenoid associated with BChl 800 absorbs some nanometers more to the red and its orientation is approximately parallel to the membrane, while the carotenoid associated with BChl 850 is oriented more or less perpendicular to the membrane. The fluorescence polarization of BChl 850 was the same for the different complexes. This indicates that the observed polarization of the fluorescence is determined by the smallest complex obtained which contains 8–10 BChl 850 molecules. The B800–850 complex isolated with LDAO thus must consist of a highly ordered array of smaller structures. On basis of these results a minimal model is proposed for the basic unit consisting of four BChl 850 and two BChl 800 and three carotenoid molecules.     

Structure and Membrane Interactions of the Antibiotic Peptide Dermadistinctin K by Multidimensional Solution and Oriented N and P Solid-State NMR Spectroscopy

DD K, a peptide first isolated from the skin secretion of the Phyllomedusa distincta frog, has been prepared by solid-phase chemical peptide synthesis and its conformation was studied in trifluoroethanol/water as well as in the presence of sodium dodecyl sulfate and dodecylphosphocholine micelles or small unilamellar vesicles. Multidimensional solution NMR spectroscopy indicates an α-helical conformation in membrane environments starting at residue 7 and extending to the C-terminal carboxyamide. Furthermore, DD K has been labeled with ¹⁵N at a single alanine position that is located within the helical core region of the sequence. When reconstituted into oriented phosphatidylcholine membranes the resulting ¹⁵N solid-state NMR spectrum shows a well-defined helix alignment parallel to the membrane surface in excellent agreement with the amphipathic character of DD K. Proton-decoupled ³¹P solid-state NMR spectroscopy indicates that the peptide creates a high level of disorder at the level of the phospholipid headgroup suggesting that DD K partitions into the bilayer where it severely disrupts membrane packing.     

Asymmetric orientation of amino groups in the α-subunit and the β-subunit of (Na+ + K+)-ATPase in tight right-side-out vesicles of basolateral membranes from outer medulla

The orientation of amino groups in the membrane in the α- and β-subunits of (Na⁺ + K⁺)-ATPase was examined by labeling with Boldon-Hunter reagent, N-succinimidyl 3-(4-hydroxy,5-[¹²⁵I]iodophenyl)propionate), in right-side-out vesicles or in open membrane fragments from the thick ascending limbs of the Henles loop of pig kidney. Sealed right-side-out vesicles of basolateral membranes were separated from open membrane fragments by centrifugation in a linear metrizamide density gradient. After labeling, (Na⁺ + K⁺)-ATPase was purified using a micro-scale version of the ATP-SDS procedure. Distribution of label was analyzed after SDS-gel electrophoresis of α-subunit, β-subunit and proteolytic fragments of α-subunit. Both the α- and the β-subunit of (Na⁺ + K⁺)-ATPase are uniformly labeled, but the distribution of labeled residues on the two membrane surfaces differs markedly. All the labeled residues in the β-subunit are located on the extracellular surface. In the α-subunit, 65–80% of modified groups are localized to the cytoplasmic surface and 20–35% to the extracellular membrane surface. Proteolytic cleavage provides evidence for the random distribution of ¹²⁵I-labeling within the α-subunit. The preservation of (Na⁺ + K⁺)-ATPase activity and the observation of distinct proteolytic cleavage patterns of the E₁- and E₂-forms of the α-subunit show that the native enzyme structure is unaffected by labeling with Bolton-Hunter reagent. Bolton-Hunter reagent was shown not to permeate into sheep erythrocytes under the conditions of the labeling experiment. The data therefore allow the conclusion that the mass distribution is asymmetric, with all the labeled amino groups in the β-subunit being on the extracellular surface, while the α-subunit exposes 2.6-fold more amino groups on the cytoplasmic than on the extracellular surface.     

Topological analysis of the pyridine nucleotide transhydrogenase of Escherichia coli using proteolytic enzymes

The pyridine nucleotide transhydrogenase of Escherichia coli has an alpha 2 beta 2 structure (alpha: Mr, 54,000; beta: Mr, 48,700). Hydropathy analysis of the amino acid sequences suggested that the 10 kDa C-terminal portion of the alpha subunit and the N-terminal 20-25 kDa region of the beta subunit are composed of transmembranous alpha-helices. The topology of these subunits in the membrane was investigated using proteolytic enzymes. Trypsin digestion of everted cytoplasmic membrane vesicles released a 43 kDa polypeptide from the alpha subunit. The beta subunit was not susceptible to trypsin digestion. However, it was digested by proteinase K in everted vesicles. Both alpha and beta subunits were not attacked by trypsin and proteinase K in right-side out membrane vesicles. The beta subunit in the solubilized enzyme was only susceptible to digestion by trypsin if the substrates NADP(H) were present. NAD(H) did not affect digestion of the beta subunit. Digestion of the beta subunit of the membrane-bound enzyme by trypsin was not induced by NADP(H) unless the membranes had been previously stripped of extrinsic proteins by detergent. It is concluded that binding of NADP(H) induces a conformational change in the transhydrogenase. The location of the trypsin cleavage sites in the sequences of the alpha and beta subunits were determined by N- and C-terminal sequencing. A model is proposed in which the N-terminal 43 kDa region of the alpha subunit and the C-terminal 30 kDa region of the beta subunit are exposed on the cytoplasmic side of the inner membrane of E. coli. Binding sites for pyridine nucleotide coenzymes in these regions were suggested by affinity chromatography on NAD-agarose columns.     

Distribution of enzymes involved in mannan synthesis in plasma membranes and mesosomal vesicles of Micrococcus lysodeikticus

The distribution of membrane-bound enzymes involved in mannan biosynthesis in plasma and mesosomal membranes of Micrococcus lysodeikticus has been investigated.Isolated mesosomal vesicles, unlike plasma membrane preparations, cannot catalyze the transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose into mannan. This appears to result from the inability of this membrane system to synthesize the carrier lipid [¹⁴C]mannosyl-l-phosphorylundecaprenol. In contrast, this is the major manno-lipid synthesized from GDP-[¹⁴C]mannose by isolated plasma membranes. The possibility that substrate inaccessibility could account for the failure to detect the enzyme in isolated mesosomal vesicles appears unlikely from the lack of activity following disruption of the vesicles with ultrasound or with surface active agents.Both membrane preparations possessed the ability to catalyse the transfer of [¹⁴C]mannose from purified [¹⁴C]mannosyl-l-phosphorylundecaprenol into mannan. Furthermore, free mannan and mannan located on both unlabeled mesosomal and unlabeled plasma membranes could act as acceptors of [¹⁴C]mannosyl units from ¹⁴C-labeled carrier lipid located in prelabeled plasma membranes. The possibility that the juxtaposition of mesosomal vesicles and enveloping plasma membrane (i.e. the mesosomal sacculus) in vivo allows mannan, located on mesosomal vesicles, to accept mannosyl units from carrier lipid located in the sacculus membrane is discussed.     

α-Enolase binds to human plasminogen on the surface of Bacillus anthracis

α-enolase of Bacillus anthracis has recently been classified as an immunodominant antigen and a potent virulence factor determinant. α-enolase (2-phospho-d-glycerate hydrolase (EC 4.2.1.11), a key glycolytic metalloenzyme catalyzes the dehydration of d-(+)-2-phosphoglyceric acid to phosphoenolpyruvate. Interaction of surface bound α-enolase with plasminogen has been incriminated in tissue invasion for pathogenesis. B. anthracis α-enolase was expressed in Escherichia coli and the recombinant enzyme was purified to homogeneity that exhibited a K_m of 3.3 mM for phosphoenolpyruvate and a V_max of 0.506 µMmin^{− 1} mg^{− 1}. B. anthracis whole cells and membrane vesicles probed with anti-enolase antibodies confirmed the surface localization of α-enolase. The specific interaction of α-enolase with human plasminogen (but not plasmin) evident from ELISA and the retardation in the native gel reinforced its role in plasminogen binding. Putative plasminogen receptors in B. anthracis other than enolase were also observed. This binding was found to be carboxypeptidase sensitive implicating the role of C-terminal lysine residues. The recombinant enolase displayed in vitro laminin binding, an important mammalian extracellular matrix protein. Plasminogen interaction conferred B. anthracis with a potential to in vitro degrade fibronectin and exhibit fibrinolytic phenotype. Therefore, by virtue of its interaction to host plasminogen and extracellular matrix proteins, α-enolase may contribute in augmenting the invasive potential of B. anthracis.     

Defective transport in S-adenosylmethionine synthetase mutants of Escherichia coli

The transport of several metabolites is decreased in mutant strains of Escherichia coli (Met K, E4 and E40), which contain decreased levels of S-adenosylmethionine synthetase. The rates and extents of uptake for lysine, leucine, methionine, and α-methylglucoside in both whole cells and membrane vesicles isolated from these mutants are 2- to 10-fold lower than in corresponding preparations from wild-type cells, although proline uptake is normal. The addition of S-adenosylmethionine to cultures of strain E40 can partially restore the rate and extent of lysine uptake. Lysine transport is lower in mutant vesicles in the presence of either d-lactate, succinate, α-hydroxylbutyrate, or NADH even though these substrates are oxidized at rates comparable to those in wild-type vesicles. This suggests that the defect is not related to the ability of vesicles to oxidize electron donors, but is very likely related to the ability of mutant vesicles to couple respiration to lysine transport. In addition, temperature-induced efflux of α-methylglucoside phosphate and dinitrophenol-induced efflux of lysine are similar in both the mutant and wild-type membranes, indicating that the barrier properties of the membrane and the activity of the lysine carrier are normal.     

Effect of alkyl alcohols on partially unfolded state of Proteinase K: Differential stability of α-helix and β-sheet rich regions of the enzyme

Proteinase K (E.C. 3.4.21.64), a serine proteinase from fungus Tritirachium album, has been used as a model system to investigate the conformational changes induced by monohydric alcohols at low pH. Proteinase K belongs to α/β class of proteins and maintains structural integrity in the range of pH 7.0–3.0. Enzyme acquires partially unfolded conformation (U_P) at pH 2.5 with lower activity, partial loss of tertiary structure and exposure of some hydrophobic patches. Proteinase K in stressed state at pH 2.5 is chosen and the conformational changes induced by alkyl alcohols (methanol/ethanol/isopropanol) are studied. At critical concentration of alcohol, conformational switch occurs in the protein structure from α/β to β-sheet driving the protein into O-state. Complete loss of tertiary contacts and proteolytic activity in O-sate emphasize the involvement of alpha regions in maintaining the active site of the enzyme. Moreover, isopropanol induced unfolding of proteinase K in U_P state occurred in two steps with the formation of β state at low alcohol concentration followed by stabilization of β state at high alcohol concentration. GuHCl and temperature induced unfolding of proteinase K in O-state (in 50% isopropanol) is non-cooperative as the transition curves are biphasic. This suggests that the structure of proteinase K in O-state has melted alpha regions and stabilized beta regions and that these differentially stabilized regions unfold sequentially. Further, the O-state of proteinase K can be attained from complete unfolded protein by the addition of 50% isopropanol. Hence the alcohol-induced O-state is different from native state or completely unfolded state and shows characteristics of the molten globule-like state. Thus, this state may be functioning as an intermediary in the folding pathway of proteinase K.