Photosystem I is the most efficient photosynthetic enzyme with structure and composition highly conserved among all oxygenic phototrophs. Cyanobacterial Photosystem I is typically associated into trimers for reasons that are still debated. Almost universally, Photosystem I contains a number of long-wavelength-absorbing ‘red’ chlorophylls (Chls), that have a sizeable effect on the excitation energy transfer and trapping. Here we present spectroscopic comparison of trimeric Photosystem I from Synechocystis PCC 6803 with a monomeric complex from the Δ psaL mutant and a ‘minimal’ monomeric complex ΔFIJL, containing only subunits A, B, C, D, E, K and M. The quantum yield of photochemistry at room temperature was the same in all complexes, demonstrating the functional robustness of this photosystem. The monomeric complexes had a reduced far-red absorption and emission equivalent to the loss of 1.5–2 red Chls emitting at 710–715 nm, whereas the longest-wavelength emission at 722 nm was not affected. The picosecond fluorescence kinetics at 77 K showed spectrally and kinetically distinct red Chls in all complexes and equilibration times of up to 50 ps. We found that the red Chls are not irreversible traps at 77 K but can still transfer excitations to the reaction centre, especially in the trimeric complexes. Structure-based Förster energy transfer calculations support the assignment of the lowest-energy state to the Chl pair B37/B38 and the trimer-specific red Chl emission to Chls A32/B7 located at the monomer–monomer interface. These intermediate-energy red Chls facilitate energy migration from the lowest-energy states to the reaction centre.

The light reactions of photosynthesis are hosted and regulated by the chloroplast thylakoid membrane (TM) — the central structural component of the photosynthetic apparatus of plants and algae. The two-dimensional and three-dimensional arrangement of the lipid–protein assemblies, aka macroorganisation, and its dynamic responses to the fluctuating physiological environment, aka flexibility, are the subject of this review. An emphasis is given on the information obtainable by spectroscopic approaches, especially circular dichroism (CD). We briefly summarise the current knowledge of the composition and three-dimensional architecture of the granal TMs in plants and the supramolecular organisation of Photosystem II and light-harvesting complex II therein. We next acquaint the non-specialist reader with the fundamentals of CD spectroscopy, recent advances such as anisotropic CD, and applications for studying the structure and macroorganisation of photosynthetic complexes and membranes. Special attention is given to the structural and functional flexibility of light-harvesting complex II in vitro as revealed by CD and fluorescence spectroscopy. We give an account of the dynamic changes in membrane macroorganisation associated with the light-adaptation of the photosynthetic apparatus and the regulation of the excitation energy flow by state transitions and non-photochemical quenching.

Caspase-9 is a critical factor in the initiation of apoptosis and as a result is tightly regulated by many mechanisms. Caspase-9 contains a Caspase Activation and Recruitment Domain (CARD), which enables caspase-9 to form a tight interaction with the apoptosome, a heptameric activating platform. The caspase-9 CARD has been thought to be principally involved in recruitment to the apoptosome, but its roles outside this interaction have yet to be uncovered. In this work, we show that the CARD is involved in physical interactions with the catalytic core of caspase-9 in the absence of the apoptosome; this interaction requires a properly formed caspase-9 active site. The active sites of caspases are composed of four extremely mobile loops. When the active-site loops are not properly ordered, the CARD and core domains of caspase-9 do not interact and behave independently, like loosely tethered beads. When the active-site loop bundle is properly ordered, the CARD domain interacts with the catalytic core, forming a single folding unit. Taken together, these findings provide mechanistic insights into a new level of caspase-9 regulation, prompting speculation that the CARD may also play a role in the recruitment or recognition of substrate.

Spermine oxidase (SMOX) is a flavin-containing enzyme that oxidizes spermine to produce spermidine, 3-aminopropanaldehyde, and hydrogen peroxide. SMOX has been shown to play key roles in inflammation and carcinogenesis; indeed, it is differentially expressed in several human cancer types. Our previous investigation has revealed that SMOX purified after heterologous expression in Escherichia coli actually consists of monomers, covalent homodimers, and other higher-order forms. All association forms oxidize spermine and, after treatment with dithiothreitol, revert to SMOX monomer. Here, we report a detailed investigation on the thermal denaturation of SMOX and its association forms in native and reducing conditions. By combining spectroscopic methods (circular dichroism, fluorescence) and thermal methods (differential scanning calorimetry), we provide new insights into the structure, the transformation, and the stability of SMOX. While the crystal structure of this protein is not available yet, experimental results are interpreted also on the basis of a novel SMOX structural model, obtained in silico exploiting the recently solved acetylspermine oxidase crystal structure. We conclude that while at least one specific intermolecular disulfide bond links two SMOX molecules to form the homodimer, the thermal denaturation profiles can be justified by the presence of at least one intramolecular disulfide bond, which also plays a critical role in the stabilization of the overall three-dimensional SMOX structure, and in particular of its flavin adenine dinucleotide-containing active site.

The ParB protein, KorB, from the RK2 plasmid is required for DNA partitioning and transcriptional repression. It acts co-operatively with other proteins, including the repressor KorA. Like many multifunctional proteins, KorB contains regions of intrinsically disordered structure, existing in a large ensemble of interconverting conformations. Using NMR spectroscopy, circular dichroism and small-angle neutron scattering, we studied KorB selectively within its binary complexes with KorA and DNA, and within the ternary KorA/KorB/DNA complex. The bound KorB protein remains disordered with a mobile C-terminal domain and no changes in the secondary structure, but increases in the radius of gyration on complex formation. Comparison of wild-type KorB with an N-terminal deletion mutant allows a model of the ensemble average distances between the domains when bound to DNA. We propose that the positive co-operativity between KorB, KorA and DNA results from conformational restriction of KorB on binding each partner, while maintaining disorder.

Two missense mutations, R33Q and L167H, of hCASQ2 (human cardiac calsequestrin), a protein segregated to the lumen of the sarcoplasmic reticulum, are linked to the autosomal recessive form of CPVT (catecholaminergic polymorphic ventricular tachycardia). The effects of these mutations on the conformational, stability and Ca 2+ sensitivity properties of hCASQ2, were investigated. Recombinant WT (wild-type) and mutant CASQ2s were purified to homogeneity and characterized by spectroscopic (CD and fluorescence) and biochemical (size-exclusion chromatography and limited proteolysis) methods at 500 and 100 mM KCl, with or without Ca 2+ at a physiological intraluminal concentration of 1 mM; Ca 2+ -induced polymerization properties were studied by turbidimetry. In the absence of Ca 2+ , mutations did not alter the conformation of monomeric CASQ2. For L167H only, at 100 mM KCl, emission fluorescence changes suggested tertiary structure alterations. Limited proteolysis showed that amino acid substitutions enhanced the conformational flexibility of CASQ2 mutants, which became more susceptible to tryptic cleavage, in the order L167H>R33Q>WT. Ca 2+ at a concentration of 1 mM amplified such differences: Ca 2+ stabilized WT CASQ2 against urea denaturation and tryptic cleavage, whereas this effect was reduced in R33Q and absent in L167H. Increasing [Ca 2+ ] induced polymerization and precipitation of R33Q, but not that of L167H, which was insensitive to Ca 2+ . Based on CASQ2 models, we propose that the Arg 33 →Gln exchange made the Ca 2+ -dependent formation of front-to-front dimers more difficult, whereas the Leu 167 →His replacement almost completely inhibited back-to-back dimer interactions. Initial molecular events of CPVT pathogenesis begin to unveil and appear to be different depending upon the specific CASQ2 mutation.

Steroidogenesis depends on the delivery of cholesterol from the outer to the inner mitochondrial membrane by StAR (steroidogenic acute regulatory protein). However, the mechanism by which StAR binds to cholesterol and its importance in cholesterol transport are under debate. According to our proposed molecular model, StAR possesses a hydrophobic cavity, which can accommodate one cholesterol molecule. In the bound form, cholesterol interacts with hydrophobic side-chains located in the C-terminal α-helix 4, thereby favouring the folding of this helix. To verify this model experimentally, we have characterized the in vitro activity, overall structure, thermodynamic stability and cholesterol-binding affinity of StAR lacking the N-terminal 62 amino acid residues (termed N-62 StAR). This mature form is biologically active and has a well-defined tertiary structure. Addition of cholesterol to N-62 StAR led to an increase in the α-helical content and T ° (melting temperature), indicating the formation of a stable complex. However, the mutation F267Q, which is located in the C-terminal helix interface lining the cholesterol-binding site, reduced the biological activity of StAR. Furthermore, the cholesterol-induced thermodynamic stability and the binding capacity of StAR were significantly diminished in the F267Q mutant. Titration of StAR with cholesterol yielded a 1:1 complex with an apparent K D of 3×10 −8 . These results support our model and indicate that StAR can readily bind to cholesterol with an apparent affinity that commensurates with monomeric cholesterol solubility in water. The proper function of the C-terminal α-helix is essential for the binding process.

GSTs (glutathione transferases) are a multifunctional group of enzymes, widely distributed and involved in cellular detoxification processes. In the xenobiotic-degrading bacterium Ochrobactrum anthropi , GST is overexpressed in the presence of toxic concentrations of aromatic compounds such as 4-chlorophenol and atrazine. We have determined the crystal structure of the GST from O. anthropi (OaGST) in complex with GSH. Like other bacterial GSTs, OaGST belongs to the Beta class and shows a similar binding pocket for GSH. However, in contrast with the structure of Proteus mirabilis GST, GSH is not covalently bound to Cys 10 , but is present in the thiolate form. In our investigation of the structural basis for GSH stabilization, we have identified a conserved network of hydrogen-bond interactions, mediated by the presence of a structural water molecule that links Ser 11 to Glu 198 . Partial disruption of this network, by mutagenesis of Ser 11 to alanine, increases the K m for GSH 15-fold and decreases the catalytic efficiency 4-fold, even though Ser 11 is not involved in GSH binding. Thermal- and chemical-induced unfolding studies point to a global effect of the mutation on the stability of the protein and to a central role of these residues in zippering the terminal helix of the C-terminal domain to the starting helix of the N-terminal domain.

Paramecium bursaria chlorella virus, a large DNA virus that replicates in unicellular Chlorella -like algae, encodes an RNA triphosphatase which is involved in the synthesis of the RNA cap structure found at the 5′ end of the viral mRNAs. The Chlorella virus RNA triphosphatase is the smallest member of the metal-dependent RNA triphosphatases that include enzymes from fungi, DNA viruses, protozoans and microsporidian parasites. In the present study, we investigated the ability of various vanadate oxoanions to inhibit the phosphohydrolase activity of the enzyme. Fluorescence spectroscopy and CD studies were used to directly monitor the binding of decavanadate to the enzyme. Moreover, competition assays show that decavanadate is a potent non-competitive inhibitor of the phosphohydrolase activity, and mutagenesis studies indicate that the binding of decavanadate does not involve amino acids located in the active site of the enzyme. In order to provide additional insight into the relationship between the enzyme structure and decavanadate binding, we correlated the effect of decavanadate binding on protein structure using both CD and guanidinium chloride-induced denaturation as structural indicators. Our data indicated that no significant modification of the overall protein architecture was occurring upon decavanadate binding. However, both fluorescence spectroscopy and CD experiments clearly revealed that the binding of decavanadate to the enzyme significantly decreased the structural stability of the enzyme. Taken together, these studies provide crucial insights into the inhibition of metal-dependent RNA triphosphatases by decavanadate.

The breast cancer susceptibility gene, BRCA1 , encodes a large nuclear phosphoprotein, the major isoform of which is 1863 amino acids in size. Structure–function studies have been largely restricted to the only two domains identified by homology searches: the RING (really interesting new gene) and BRCT (BRCA1 C-terminus) domains. However, we have recently reported the identification of a large central soluble region of BRCA1 (residues 230–534) that binds specifically to four-way junction DNA, a property that potentially facilitates its role in the repair of DNA lesions by homologous recombination. We have now used a combination of limited proteolysis and extension cloning to identify more accurately the DNA-binding region of BRCA1. Limited trypsinolysis of BRCA1-(230–534) resulted in the production of a soluble domain identified as residues 230–339. However, after cloning, expression and purification of this region, studies revealed that it was unable to bind to four-way junctions, suggesting that the DNA-binding activity, in part, resides within residues 340–534. A series of fragments extending from residue 340 were produced, and each was tested for its ability to bind to four-way junction DNA in gel retardation assays. In these experiments, residues 340–554 of BRCA1 were identified as the minimal DNA-binding region. We then went on to characterize the conformation of this region using CD spectroscopy and analytical centrifugation.

In previous studies, we showed that the chemical and dynamic properties of fish and mouse MTs (metallothioneins) present a number of distinctive differences linked to their primary structures, and that phylogenetic relationships of mammal and fish MTs correlate with their three-dimensional structures. The different behaviours of MTs may also be linked to the interaction between their two domains. In the present study, we have compared the physicochemical properties of the isolated recombinant domains constituting Notothenia coriiceps and mouse MTs, and compared them with those of the corresponding whole MTs. NMR spectra of the separated domains of N. coriiceps are almost superimposable on those of the parent MT, suggesting an apparent lack of interaction between the two domains in the protein. However, certain dynamic and physicochemical features of the isolated domains are unlike those of the whole protein. In particular, the temperature-induced changes in the chiroptical properties, thiol reactivity of the Zn-MT domains and the Zn 2+ /Cd 2+ rate of exchange are different for the two domains and with respect to the whole protein. Taken together, these results provide a strong argument in favour of the interaction of the two domains in the MT molecule, in spite of the elusive evidence provided by the structural analyses.

We have established a novel assay method based on circular dichroism that can be used for the kinetic study of the activity of amino acid racemases, such as ALR (alanine racemase). Although an enzyme-coupled assay method has been used to measure racemase activity, the CD method is superior to the enzyme assay because it can accurately determine the immediate changes of an enantiomer on racemization between its L - and D -forms. The enzyme-coupled assay requires D -amino acid oxidase, which is inactivated by an inhibitor of ALR, D -cycloserine. This indicates that the inhibitory kinetic study for ALR with D -cycloserine by the enzyme-coupled assay method is restricted to the analysis of only the reaction resulting in the formation of L -Ala from D -Ala. However, since the CD assay does not require the coupled enzyme, it can be used to comprehensively evaluate the reactions that result in the formation both of D -Ala from L -Ala and of L -Ala from D -Ala at several substrate concentrations. Streptomyces ALR also catalyses the formation of D -Ser from L -Ser and of L -Ser from D -Ser, but the catalytic constants ( k cat ) are 4- and 10-fold lower than those for the formation of D -Ala from L -Ala and of L -Ala from D -Ala respectively.

TraA is the DNA relaxase encoded by the broad-host-range Grampositive plasmid pIP501. It is the second relaxase to be characterized from plasmids originating from Gram-positive organisms. Full-length TraA (654 amino acids) and the N-terminal domain (246 amino acids), termed TraAN 246 , were expressed as 6×His-tagged fusions and purified. Small-angle X-ray scattering and chemical cross-linking proved that TraAN 246 and TraA form dimers in solution. Both proteins revealed oriT pIP501 (origin of transfer of pIP501) cleavage activity on supercoiled plasmid DNA in vitro . oriT binding was demonstrated by electrophoretic mobility shift assays. Radiolabelled oligonucleotides covering different parts of oriT pIP501 were subjected to binding with TraA and TraAN 246 . The K D of the protein–DNA complex encompassing the inverted repeat, the nick site and an additional 7 bases was found to be 55 nM for TraA and 26 nM for TraAN 246 . The unfolding of both protein constructs was monitored by measuring the change in the CD signal at 220 nm upon temperature change. The unfolding transition of both proteins occurred at approx. 42 °C. CD spectra measured at 20 °C showed 30% α-helix and 13% β-sheet for TraA, and 27% α-helix and 18% β-sheet content for the truncated protein. Upon DNA binding, an enhanced secondary structure content and increased thermal stability were observed for the TraAN 246 protein, suggesting an induced-fit mechanism for the formation of the specific relaxase– oriT complex.

The functional role of three conserved amino acid residues in Proteus mirabilis glutathione S-transferase B1-1 (PmGST B1-1) has been investigated by site-directed mutagenesis. Crystallographic analyses indicated that Glu 65 , Ser 103 and Glu 104 are in hydrogen-bonding distance of the N-terminal amino group of the γ-glutamyl moiety of the co-substrate, GSH. Glu 65 was mutated to either aspartic acid or leucine, and Ser 103 and Glu 104 were both mutated to alanine. Glu 65 mutants (Glu 65 →Asp and Glu 65 →Leu) lost all enzyme activity, and a drastic decrease in catalytic efficiency was observed for Ser 103 →Ala and Glu 104 →Ala mutants toward both 1-chloro-2,4-dinitrobenzene and GSH. On the other hand, all mutants displayed similar intrinsic fluorescence, CD spectra and thermal stability, indicating that the mutations did not affect the structural integrity of the enzyme. Taken together, these results indicate that Ser 103 and Glu 104 are significantly involved in the interaction with GSH at the active site of PmGST B1-1, whereas Glu 65 is crucial for catalysis.

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K + channels, including the voltage-gated Shaker B subtype. In the present study, we have investigated over 80h: (1) the time-course of folding of synthetic MTX (sMTX) by CD analysis; (2) the kinetics of disulphide bridge formation by MS; and (3) the potency of MTX in blocking Shaker B currents during the combined process of its in vitro folding and oxidation. From the CD data, we show that stable secondary structures of sMTX evolve sequentially over time, with the appearance of the α-helix within 5h, followed by the formation of the β-sheet within 22h. Using MS analysis, the sMTX intermediates were also found to appear sequentially from the least (one-disulphide-bridged sMTX) to the most oxidized species (native-like, four-disulphide-bridged sMTX). The time course of formation of secondary structures coincides mainly with the occurrence of one-disulphide-bridged sMTX for the α-helix and two- or three-disulphide-bridged sMTX for the β-sheet. On-line electrophysiological recordings, which measure sMTX blocking efficacy on K + currents during its folding and oxidation, were performed on Shaker B channels expressed in Xenopus oocytes. Unexpectedly, the results demonstrate that sMTX is highly potent at the initial stage of oxidation, whereas its blocking activity can be transiently and dramatically reduced at later stages during the course of folding/oxidation before it reaches full bioactivity. These data suggest that formation of disulphide bridges can both physically stabilize and alter the bioactive three-dimensional structure of sMTX.

Interaction between two αβ half-receptors within the (αβ) 2 holoreceptor complex is required for insulin binding with high affinity and for insulin-triggered changes of size and shape. To understand the underlying structure–function relationship, two truncated receptor constructs have been characterized. Reduction in the Stokes radius and increase in the sedimentation coefficient, which are characteristic for wild-type receptors, were entirely lacking for the recombinant human insulin receptor (HIR) ectodomain (HIR-ED). Stokes radii of about 5.8nm and sedimentation coefficients of 10.2S were found for both insulin-bound and free HIR-EDs. However, attaching the membrane anchors to the ectodomain, as with the recombinant membrane-anchored ectodomain (HIR-MAED) construct, was sufficient to restore not only high-affinity hormone binding but also the marked insulin-inducible alterations in hydrodynamic properties. The Stokes radii of HIR-MAED complexes, as assessed by non-denaturing PAGE, decreased upon insulin binding from 9.5nm to 7.9nm. In parallel, the sedimentation coefficient was increased from 9.0S to 9.8S. CD and fluorescence spectroscopy of HIR-MAED revealed only minor insulin-induced changes in the secondary structure. Similarity with wild-type receptors has also been demonstrated by the differential insertion of insulin-bound and free HIR-MAED complexes into artificial bilayer membranes of Triton X-114. The results are consistent with a model of receptor function that ensures a global insulin-triggered reorientation of subdomains within the ectodomain moieties while the secondary structure is essentially retained. For the rearrangement of such subdomains, the transmembrane anchors confer essential structural constraints on the receptor ectodomain.

Fish and mammalian metallothioneins (MTs) differ in the amino acid residues placed between their conserved cysteines. We have expressed the MT of an Antarctic fish, Notothenia coriiceps , and characterized it by means of multinuclear NMR spectroscopy. Overall, the architecture of the fish MT is very similar to that of mammalian MTs. However, NMR spectroscopy shows that the dynamic behaviour of the two domains is markedly different. With the aid of absorption and CD spectroscopies, we studied the conformational and electronic features of fish and mouse recombinant Cd-MT and the changes produced in these proteins by heating. When the temperature was increased from 20 to 90°C, the Cd-thiolate chromophore absorbance at 254nm of mouse MT was not modified up to 60°C, whereas the absorbance of fish MT decreased significantly starting from 30°C. The CD spectra also changed quite considerably with temperature, with a gradual decrease of the positive band at 260nm that was more pronounced for fish than for mouse MT. The differential effect of temperature on fish and mouse MTs may reflect a different stability of metal-thiolate clusters of the two proteins. Such a conclusion is also corroborated by results showing differences in metal mobility between fish and mouse Zn-MT.

Chemical modification experiments with tetranitromethane (TNM) have been used to investigate the role of tyrosine residues in the formation of the complex between PpL (the single Ig-binding domain of protein L, isolated from P. magnus strain 3316) and the kappa light chain (κ-chain). Reaction of PpL with TNM causes the modification of 1.9 equiv. of tyrosine (Tyr 51 and Tyr 53 ) and results in an approx. 140-fold decrease in affinity for human IgG. Similar experiments with mutated PpL proteins suggest that nitration predominantly inactivates the protein by modification of Tyr 53 . Reduction of the nitrotyrosine groups to aminotyrosine by incubation with sodium hydrosulphite does not restore high affinity for IgG. Modification of κ-chain by TNM resulted in the nitration of 3.1±0.09 tyrosine residues. When the PpLŐκ-chain complex was incubated with TNM, 4.1±0.04 tyrosine residues were nitrated, indicating that one tyrosine residue previously modified by the reagent was protected from TNM when the proteins are in complex with each other. The K d for the equilibrium between PpL, human IgG and their complex has been shown by ELISA to be 112±20nM. A similar value (153±33nM) was obtained for the complex formed between IgG and the Tyr 64 → Trp mutant (Y64W). However, the K d values for the equilibria involving the PpL mutants Y53F and Y53F,Y64W were found to be 3.2±0.2 and 4.6±1 µ M respectively. These suggest that the phenol group of Tyr 53 in PpL is important to the stability of the PpLŐκ-chain complex.

We have reported previously on the expression of recombinant human type X collagen (hrColX) in HEK 293 and HT 1080 cells by using the eukaryotic expression vector pCMVsis (in which CMV stands for cytomegalovirus). Several stably transfected clones secreted full-length triple-helical hrColX molecules in large amounts, but the secreted collagen was underhydroxylated, with a hydroxyproline-to-proline ratio of 0.25 and a melting temperature ( T m ) of 31°C. By comparison, native chicken type X procollagen has a T m of 46°C. To stabilize the triple helix of hrColX, an hrColX-expressing clone (A6/16) was co-transfected with both α and β subunits of human prolyl 4-hydroxylase. Clones were selected that secreted proα1(X) collagen chains with an apparent molecular mass of 75kDa and an increased hydroxyproline-to-proline ratio of close to 0.5. As a result of enhanced prolyl hydroxylation, the T m of the hrColX was increased to 41°C as measured by CD analysis at various temperatures. The CD spectra indicated a minimum ellipticity at 198nm and a peak at 225nm at 20°C, confirming the presence of a triple helix. The same T m of 41°C was measured for the triple-helical core fragments of hrColX of 60Ő65kDa that were retained after brief digestion with chymotrypsin/trypsin at increasing temperatures. This shows that the human cell line HEK-293 is suitable for the simultaneous expression of three genes and the stable production of substantial amounts of recombinant, fully hydroxylated type X collagen over several years.

An 18-residue peptide, corresponding to the minimum sequence of the N-terminal zinc-finger domain in the nucleocapsid of human T-lymphotrophic virus type I, was synthesized by a solid-phase method and fully characterized. Its ability to complex metal ions (Co 2+ and Zn 2+ ) was clearly established by UV–visible spectroscopy and MS. The stability of these complexes was investigated by an original method with HPLC chromatography. Our results show that, even in the presence of air, the Zn 2+ complex is highly stable. In contrast, the Co 2+ complex undergoes a relatively fast degradation due to an intramolecular oxidation leading to the formation of a disulphide bridge between two cysteine residues. The 1 H-NMR analysis indicates that Zn 2+ binds to the Nδ atom of the histidine residue rather than to the Nε atom. Two-dimensional NMR techniques were used to determine the solution structure of the zinc-finger, illustrated by the existence of turns in the overall conformation.

Hsp47, a 47kDa heat shock protein whose expression level parallels that of collagen, has been regarded as a collagen-specific molecular chaperone. Studies from other laboratories have established the association of Hsp47 with the nascent as well as the triple-helical procollagen molecule in the endoplasmic reticulum and its dissociation from procollagen in the Golgi. One of several roles suggested for Hsp47 in collagen biosynthesis is the prevention of aggregation of procollagen in the endoplasmic reticulum. However, no experimental evidence has been available to verify this suggestion. In the present study we have followed the aggregation of mature triple-helical collagen molecules into fibrils by using turbidimetric measurements in the absence and presence of Hsp47. In the pH range 6–7, fibril formation of type I collagen, as monitored by turbidimetry, proceeds with a lag of approx. 10min and levels off by approx. 60min. The addition of Hsp47 at pH 7 effectively inhibits fibril formation at and above a 1:1 molar ratio of Hsp47 to triple-helical collagen. This inhibition is markedly pH-dependent, being significantly diminished at pH 6. CD and fluorescence spectral data of Hsp47 in the pH range 4.2–7.4 reveal a significant alteration in its structure at pH values below 6.2, with a decrease in α-helix and an increase in β-structure. This conformational change is likely to be the basis of the decreased binding of Hsp47 to collagen in vitro at pH 6.3 as well as its inability to inhibit collagen fibril formation at this pH. Our results also provide a functional assay for Hsp47 that can be used in studies on collagen and Hsp47 interactions.

The interaction of apolipoprotein H (Apo H) with lipid membrane has been considered to be a basic mechanism for the biological function of the protein. Previous reports have demonstrated that Apo H can interact only with membranes containing anionic phospholipids. Here we study the membrane-induced conformational change of Apo H by CD spectroscopy with two different model systems: anionic-phospholipid-containing liposomes [such as 1,2-dimyristoyl- sn -glycero-3-phosphoglycerol (DMPG) and cardiolipin], and the water/methanol mixtures at moderately low pH, which mimic the micro-physicochemical environment near the membrane surface. It is found that Apo H undergoes a remarkable conformational change on interaction with liposomes containing anionic phospholipid. To interact with liposomes containing DMPG, there is a 6.8% increase in α-helix in the secondary structures; in liposomes containing cardiolipin, however, there is a 12.6% increase in α-helix and a 9% decrease in β-sheet. The similar conformation change in Apo H can be induced by treatment with an appropriate mixture of water/methanol. The results indicate that the association of Apo H with membrane is correlated with a certain conformational change in the secondary structure of the protein.

Although neither the physiological function nor the mechanism of action of sterol carrier protein 2 (SCP 2 ) is yet completely clear, it is thought that SCP 2 interacts with membranes to elicit its biological effects. The results presented here show that the SCP 2 N-terminus, composed of two amphipathic α-helices, interacted preferentially with highly curved but not lower-curvature membranes containing anionic phospholipid. CD spectra of SCP 2 showed up to 1.2-fold increased α-helical content, on the interaction of SCP 2 with small unilamellar vesicles (SUV) (median radius 10-14 nm) but less with large unilamellar vesicles (LUV) (median radius 52-60 nm). Although enhanced interaction with the SUV membranes was due in part to the radius of curvature and to the greater exposure of acidic phospholipid in the outer leaflet of the bilayer, simply increasing the molar percentage of acidic phospholipid in the LUV membranes had much less effect on SCP 2 binding. A similar preferential interaction was observed with highly curved SUV as opposed to LUV for the SCP 2 N-terminal peptide 1-32 SCP 2 as well as structurally modified peptides in the order 1-32 SCP 2 = 10-32 SCP 2 > 1-24 SCP 2 ≫ 1-E20-32 SCP 2 . The CD results were confirmed with an independent filtration binding assay, which showed that SCP 2 bound 5-fold more to SUV than LUV, whereas its N-terminal peptides bound up to 4-fold better in the order 1-32 SCP 2 = 10-32 SCP 2 > 1-24 SCP 2 > 1-E20-32 SCP 2 . Finally, cholesterol potentiated the binding of SCP 2 and N-terminal peptides to anionic-phospholipid-containing SUV but not LUV. These findings were consistent with the SCP 2 N-terminus being a membrane-binding domain that was highly dependent on membrane surface curvature as well as on lipid composition.

We previously showed that bovine apolipoprotein A-II (apoA-II) had antimicrobial activity against Escherichia coli and the yeast Saccharomyces cerevisiae in PBS. We have characterized here the active domain of apoA-II using synthetic peptides. A peptide corresponding to C-terminal residues Leu 49 -Thr 76 exhibited significant antimicrobial activity against E. coli in PBS, but not against S. cerevisiae . Experiments using amino-acid-substituted peptides indicated that the residues Phe 52 -Phe 53 -Lys 54 -Lys 55 are required for the activity. Peptide Leu 49 -Thr 76 induced the release of calcein trapped inside the vesicles whose lipid composition resembles that of E. coli membrane, suggesting that peptide Leu 49 -Thr 76 can destabilize the E. coli membrane. CD measurements showed that the α-helicity of peptide Leu 49 -Thr 76 increased from 3.5 to 36% by addition of the vesicles. When E. coli cells were incubated with peptide Leu 49 -Thr 76 , some proteins were released to the external medium, probably owing to membrane destabilization caused by the peptide. In electron micrographs of E. coli cells treated with peptide Leu 49 -Thr 76 , transparent nucleoids and granulated cytoplasm were observed. Amino acid substitutions, Phe 52 Phe 53 → AlaAla (Phe 52,53 → Ala) in peptide Leu 49 -Thr 76 caused the loss of antimicrobial activity against E. coli, although protein-releasing activity was retained. Electron micrographs of the cells treated with peptide Leu 49 -Thr 76 (Phe 52,53 → Ala) revealed morphological change only at the nucleoids. Therefore peptide Leu 49 -Thr 76 appears to primarily target the cytoplasm rather than the membrane of E. coli cells.

The single-stranded DNA sequence d(GT 5 G 4 CT 4 C) occurs close to the origin of replication within the intergenic region of the viral strand of bacteriophage fd. The RNA analogue of this sequence r(GU 5 G 4 CU 4 C) forms part of the untranslated leader sequence of the gene 2 mRNA and is specifically bound by the fd gene 5 protein in its role as a translational repressor. The structure of these sequences is likely to have an important role in the control of both DNA replication and RNA translation in the phage. We show that this 16 nt sequence, in both a DNA and an RNA context, can exist in a structured and unstructured form as determined by high-resolution gel filtration and non-denaturing gel electrophoresis. The CD spectrum of the structured form is characteristic of parallel guanine tetraplexes. The structured form of the DNA sequence melts at approx. 47 °C in the presence of Na + ions but the structure is stabilized up to 75 °C in the presence of K + ions. The RNA structure is more stable than the equivalent DNA structure (melting temperature approx. 62 °C), and its stability is further enhanced in the presence of K + ions. Two of the central guanine residues are fully protected from cleavage as determined by dimethyl sulphate protection experiments, whereas methylation interference studies show that methylation of any of the four central guanine residues inhibits structure formation. Our results demonstrate that the structured form of the nucleic acid is mediated through the formation of a guanine-tetraplex core region, in RNA this might be further stabilized by the presence of weaker uracil quartets.

In order to investigate further the determinants of protein stability, four mutants of thioredoxin from Bacillus acidocaldarius were designed: K18G, R82E, K18G/R82E, and D102X, in which the last four amino acids were deleted. The mutants were constructed on the basis of molecular dynamic studies and the prediction of the structure of thioredoxin from B. acidocaldarius , performed by a comparative molecular modelling technique using Escherichia coli thioredoxin as the reference protein. The mutants obtained by PCR strategy were expressed in E. coli and then characterized. CD spectroscopy, spectrofluorimetry and thermodynamic comparative studies permitted comparison of the relative physicochemical behaviour of the four proteins with that of the wild-type protein. As predicted for the molecular dynamic analysis at 500 K in vacuo , the wild-type structure was more stable than that of the mutants; in fact the T m of the four proteins showed a decrease of about 15 °C for the double and the truncated mutants, and a decrease of about 12 °C for the single mutants. A difference in the resistance of the proteins to denaturants such as guanidine HCl and urea was revealed; the wild-type protein always proved to be the most resistant. The results obtained show the importance of hydrogen bonds and ion pairs in determining protein stability and confirm that simulation methods are able to direct protein engineering in site-directed mutagenesis.