Different molten globule-like folding intermediates of hen egg white lysozyme induced by high pH and tertiary butanol

We have provided evidence that hen egg white lysozyme (HEWL) existed in alpha helical and beta structure dominated molten globule (MG) states at high pH and in the presence of tertiary butanol, respectively. Circular dichroism (CD), intrinsic fluorescence, ANS binding and acrylamide-induced fluorescence quenching techniques have been used to investigate alkali-induced unfolding of HEWL and the effect of tertiary butanol on the alkaline-induced state. At pH 12.75, HEWL existed as molten globule like intermediate. The observed MG-like intermediate was characterized by (i) retention of 77% of the native secondary structure, (ii) enhanced binding of ANS (approximately 5 times) compared to native and completely unfolded state, (iii) loss of the tertiary structure as indicated by the tertiary structural probes (near-UV, CD and Intrinsic fluorescence) and (iv) acrylamide quenching studies showed that MG state has compactness intermediate between native and completely unfolded states. Moreover, structural properties of the protein at isoelectric point (pI) and denatured states have also been described. We have also shown that in the presence of 45% tertiary butanol (t-butanol), HEWL at pH 7.0 and 11.0 (pI 11.0) existed in helical structure without much affecting tertiary structure. Interestingly, MG state of HEWL at pH 12.7 transformed into another MG state (MG2) at 20% t-butanol (v/v), in which secondary structure is mainly beta sheets. On further increasing the t-butanol concentration alpha helix was found to reform. We have proposed that formation of both alpha helical and beta sheet dominated intermediate may be possible in the folding pathway of alpha + beta protein.     

Structure and stability of human thyroxine-binding globulin.

The secondary and tertiary structure of human plasma thyroxine-binding globulin (TBG) was investigated by circular dichroism and fluorescence properties. The relaxation time of TBG indicated that it is a compact, symmetric molecule. It was calculated from the far ultraviolet CD spectrum that about one-half of the peptide groups are equally distributed in alpha helical and beta structures. In the near ultraviolet, the CD spectrum of TBG was modified when thyroxine was bound. TBG was stable at temperatures below 50 degrees at pH 9 and below 35 degrees at pH 10.5. Below pH 5 tryptophanyl fluorescence revealed a molecular transition which followed first order kinetics. The transition resulted in an irreversible loss of binding of the hormone. Acidification to pH 3.4 produced only a minor change in the CD spectrum, in which some of the alpha helical peptides were converted to beta structure.     

Circular dichroism studies on the alpha-D-galactopyranosyl binding lectin isolated from the seeds of Bandeiraea simplicifolia.

The conformation of the alpha-D-galactopyranosyl binding lectin isolated from Bandeiraea simplicifolia seeds has been investigated over a broad range of pH in the presence of various solvents by circular dichroism (CD) spectroscopy in the region 200-300 nm. Analyses of the spectra obtained on the native protein show the lectin to contain a considerable proportion of beta structure (30-40%). The native conformation was found to be largely insensitive to changes in pH, but was influenced by sodium dodecyl sulfate or trifluoroethanol. Alterations in conformation in the presence of these agents were reflected in the CD spectra and show the presence of alpha helix under these conditions. These changes in conformation are accompanied by a loss in polysaccharide-precipitating activity. The protein is irreversibly denatured in 8 M urea. Neither removal of the intrinsic calcium ions from the protein nor addition of methyl alpha-D-galactopyranoside induces any appreciable change in the CD spectra of the protein although the former treatment abolishes the polysaccharide-precipitating capacity of the lectin. The conformational data obtained in the present study are compared with data available from conformational studies of other lectins and leads to the hypothesis that most lectins probably contain beta structure as the predominant conformational feature.     

Conformational studies on the beta subunits of human hemoglobin and their arginyl-COOH peptides.

The beta subunits of hemoglobin upon alkylation of the cysteinyl residues with iodoacetamide showed a sedimentation velocity with an S20w, near 1.8 as for monomeric subunits. They reacted with alpha chains to give a tetrameric hemoglobin with a sedimentation constant near 4.4. Their CD spectrum was indistinguishable from that of untreated beta chains below 270 nm, otherwise they showed some deviation that became pronounced in the Soret region, where the optical activity of the alkylated subunits was definitely lower than that of the native subunits. Upon removal of the heme the apo-beta subunits showed a decreased optical activity in the far-uv region of the spectrum indicating a substantial loss of helical content. Their sedimentation behavior was consistent with the presence of large aggregates, which dissociates into monomers upon reconstitution with cyanoheme. The apo-beta subunits could be renatured from 6 M guanidine hydrochloride. They showed a stoichiometric reaction with heme in the molar ratio 1:1. Upon reconstitution with the heme their optical activity became similar to that of the native beta chains in the far-uv region of the spectrum, but remained lower in the near-uv and Soret regions. After acylation of the lysyl residues with citraconic anhydride the apo-beta subunits were digested with trypsin and the arginyl-COOH peptides beta(1-30), beta(31-40), beta(41-104), and beta(105-146) were separated by gel chromatography. With the exception of the peptide beta/105-146), which was insoluble at neutral pH, the sedimentation behavior of the other peptides showed the presence of small polymers. The sedimentation behavior of the peptide beta(31-40) was not tested. The percentage of alpha helix, beta conformation, and of random coil (or unordered structure) of the various proteins and peptides was measured fitting their CD spectra in the far-uv region with the parameter published by Y.H. Chen et al. ((1974), Biochemistry 13, 3350) and by N. Greenfield and G.D. Fasman ((1969), Biochemistry 8, 4108). In this way the helical content of the native and reconstituted alkylated beta subunits appeared to be near 76%, a value very near to that present in the same subunits in the hemoglobin crystal. The helical content of the apo-beta subunits in 0.04 M borate buffer at pH 9.6 decreased to a value near 45%. The helical content of the isolated peptides in electrolyte solutions was in any case near 10% indicating an almost complete loss of the structure that they have in the hemoglobin crystal. Cyanoheme reacted with the peptide beta(41-104), however, the reaction was not stoichiometric indicating a low affinity of the heme for the peptide. With the exception of the peptide beta(31-104), all of the other peptides recovered some of their helical structure when dissolved in 50% methanol. Notably also the apo-beta subunits did so suggesting that the loss of structure upon the removal of the heme could be in part due to the exposure of the heme pocket to water.     

Structural characterization of peptide fragments from hCD81-LEL.

The solution conformation of two peptides [1: PSGSNIISNLFKED; 2: GSSTLTALTTSVLKNNL] from human CD81 (hCD81) large extra-cellular loop (LEL) with known importance in the hepatitis C virus glycoprotein E2 (HCV-E2) binding interaction was characterized using circular dichroism spectroscopy. In addition, the solution structure of peptide 1 that contains a phenylalanine residue (F186 in hCD81) known to be critical in the binding interaction with HCV-E2 was determined using 1D and 2D 1H NMR spectroscopy. Both peptides are unstructured in water but begin forming significant helical conformation following the addition of 20% or more trifluoroethanol (v/v), a result consistent with their alpha-helical conformation found in the native protein. The CD data recorded as a function of pH and NaCl concentration are consistent with stabilization of the helical structure from electrostatic forces for both peptides. Peptide 1 is able to block the binding interaction of recombinant HCV-E2 (rHCV-E2) to hCD81 expressed on Molt-4 T cells at high concentrations (3.5 mM), a low affinity that we attributed to the random coil structure in water.     

Conformational analysis of peptide fragments derived from the peripheral subunit-binding domain from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus: evidence for nonrandom structure in the unfolded state

There is currently a great deal of interest in the early events in protein folding. Two issues that have generated particular interest are the nature of the unfolded state under native conditions and the role of local interactions in folding. Here, we report the results of a study of a set of peptides derived from a small two-helix protein, the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex. Five peptides of overlapping sequence were prepared, including sequences corresponding to each of the helices and to the region connecting them. The peptides were characterized by CD and, where possible, nmr. A peptide corresponding to the second helix is between 12 and 17% helical at neutral pH. CD also indicates a lower percentage of helical structure in the peptide corresponding to the first alpha-helix, although the values of the alpha-proton chemical shifts suggest some preference for nonrandom structure. Peptides corresponding to the interhelical loop, which in the full domain contains two overlapping beta-turns and a 5-residue 3(10)-helix, are less structured. There is no significant change in the helicity of any of these peptides with pH. To test for fragment complementation, CD spectra of the two peptides derived from each helix and the long connecting peptide were compared to the spectra of each possible pair, as well as to a mixture containing all three. No increase in structure was observed. We complement our peptide studies by characterizing a point mutant, D34V, which disrupts a critical hydrogen bonding network. This mutant is unable to fold and provides a useful model of the denatured state. The mutant is between 9 and 16% helical as judged by CD. The modest amount of helical structure formed in some of the peptide fragments and in the point mutant suggests that the denatured state of the peripheral subunit binding domain is not completely unstructured. This may contribute to the very rapid folding observed for the intact protein.     

Stabilization of helical structure in two 17-residue amphipathic analogues of the C-terminal peptide of cytochrome C

The conformations of two 17-residue peptide analogues derived from the C-terminal sequence of pigeon cytochrome c (native sequence = KAERADLIAYLKQATAK) were examined in aqueous and lipid environments by CD spectroscopy. The two analogues, KKLLKKLIAYLKQATAK (K peptide) and EELLEELIAYLKQATAK (E peptide), were made amphipathic with respect to helical segregation by substituting a 6-residue sequence at the N-terminus of the native peptide. Their structures were compared to the native peptide under aqueous conditions of varying pH and temperature, and in the presence of liposomes composed of phosphatidylcholine and phosphatidylserine in the ratio of 9:1. The results indicated that the native peptide remains unstructured under all the conditions examined even though this region of the native molecule is surface exposed and helical. The E peptide, however, was helical under aqueous conditions at 25 degrees C from pH 2-10 with a maximum helicity at pH 4 (54% helix from analysis of CD data). The ellipticity of the E peptide at pH 4 and 8 was concentration dependent, indicating an aggregation phenomenon. In studies in which the CD spectrum was measured at different temperatures, the E peptide became more helical at lower temperatures at pH 4 but not at pH 8. Upon interaction with a lipid membrane in the form of liposomes, there appeared to be a slight destabilization in the structure of the E peptide. The K peptide in an aqueous environment behaved like the native peptide in that it was structureless at all pHs and temperatures examined. In the presence of liposomes, however, this peptide had a high helical content (75% helix from analysis of CD data). These findings suggest that while stabilization of the helix dipole with negative charges at the N-terminus are important in inducing helical conformation in the E peptide, hydrophobic interactions created during aggregation appear to provide the principal stabilizing force. The results with the K peptide demonstrate that the positive N-terminal sequence of this peptide is able to interact with the negatively charged head groups in the phospholipid membrane in such a fashion as to stabilize a helical structure that is not apparent in an aqueous environment alone.     

Lecithin:cholesterol acyltransferase activation by synthetic amphipathic peptides

The amphipathic helical theory of Segrest and colleagues (FEBS Lett.:38:247-253, 1974) proposes that the lipid-binding segments of serum apolipoproteins are in an alpha helical conformation. Furthermore the helices have a hydrophobic face and a hydrophilic face with a specific distribution of positively and negatively charged residues. The importance of the pattern of the charged residues in the lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation by the segments is still debated. We designed a 30-residue peptide, GALA, which in the alpha helical conformation has a hydrophilic face composed of glutamic acid residues (Sabbarao et al.: Biochemistry 26:2964-2972, 1987). GALA behaves like the serum apolipoproteins in its interaction with dimyristoylphosphatidylcholine (DMPC) at neutral pH; the amino terminal tryptophan of GALA undergoes a blue shift in its fluorescence emission spectrum, and the circular dichroism (CD) spectrum indicates that GALA acquires alpha helical structure in the presence of DMPC. A DMPC-GALA:19/1 (molar ratio) complex can be isolated by gel-permeation chromatography. This complex has a discoidal structure with the approximate dimensions of 44-A edge thickness and a 170- to 350-A diameter. GALA activates LCAT with DMPC but not with unsaturated phospholipids as the substrate. The apparent partition coefficient of GALA into DMPC vesicles is 100-fold larger than into egg phosphatidylcholine vesicles. The interaction of GALA with unsaturated lipids at neutral pH is so weak that no detectable change in the spectroscopic properties of GALA or the structure of the liposomes can be detected under the conditions used here. The sequence of GALA differs from previously studied model Apo A1 peptides by the absence of positively charged residues on the hydrophilic face. This indicates that positive charges in Apo A1-like peptides are not required in order to form discoidal structures with saturated phospholipids or to activate LCAT with such lipid substrates.     

Localization of sequence segments forming a kappa-bungarotoxin-binding site on the alpha 3 neuronal nicotinic receptor

To identify the sequence segments of the alpha 3 subunit of the neuronal nicotinic acetylcholine receptor (N-nAChR) forming the binding site for the cholinergic antagonist kappa-bungarotoxin (kappa-BGT), overlapping peptides corresponding to the complete alpha 3 sequence were tested for their ability to bind 125I-labeled kappa-BGT. Two peptides located within the N-terminal extracellular domain specifically bound kappa-BGT in a solid phase assay, i.e. peptide N alpha(3)51-70 with a Kd approximately 300 nM and peptide N alpha(3)1-18 with slightly lower affinity (Kd approximately 500 nM). Preincubation of 125I-kappa-BGT with peptides N alpha(3)51-70 or N alpha(3)1-18 resulted in greater than 90% inhibition of kappa-125I-BGT binding to native N-nAChR expressed on the neuronal cell line PC12. Under the same conditions, two additional peptides, N alpha(3)180-199 and N alpha(3)183-201, were found to inhibit kappa-125I-BGT binding to PC12 by approximately 50%. These latter peptides represent sequences that are homologous to those shown previously to bind alpha-bungarotoxin. Peptide N alpha(3)51-70 (400 microM) also reduced by approximately 4-fold the observed rate of association of kappa-BGT to PC12 cells. The results of these experiments identify sequence segments of the alpha 3 subunit which are likely to interact with kappa-BGT and may indicate the relative contribution that these segments make in the formation of the high affinity kappa-BGT-binding site of this N-nAChR subtype.     

Structures and mode of membrane interaction of a short alpha helical lytic peptide and its diastereomer determined by NMR, FTIR, and fluorescence spectroscopy.

The interaction of many lytic cationic antimicrobial peptides with their target cells involves electrostatic interactions, hydrophobic effects, and the formation of amphipathic secondary structures, such as alpha helices or beta sheets. We have shown in previous studies that incorporating approximately 30%d-amino acids into a short alpha helical lytic peptide composed of leucine and lysine preserved the antimicrobial activity of the parent peptide, while the hemolytic activity was abolished. However, the mechanisms underlying the unique structural features induced by incorporating d-amino acids that enable short diastereomeric antimicrobial peptides to preserve membrane binding and lytic capabilities remain unknown. In this study, we analyze in detail the structures of a model amphipathic alpha helical cytolytic peptide KLLLKWLL KLLK-NH2 and its diastereomeric analog and their interactions with zwitterionic and negatively charged membranes. Calculations based on high-resolution NMR experiments in dodecylphosphocholine (DPCho) and sodium dodecyl sulfate (SDS) micelles yield three-dimensional structures of both peptides. Structural analysis reveals that the peptides have an amphipathic organization within both membranes. Specifically, the alpha helical structure of the L-type peptide causes orientation of the hydrophobic and polar amino acids onto separate surfaces, allowing interactions with both the hydrophobic core of the membrane and the polar head group region. Significantly, despite the absence of helical structures, the diastereomer peptide analog exhibits similar segregation between the polar and hydrophobic surfaces. Further insight into the membrane-binding properties of the peptides and their depth of penetration into the lipid bilayer has been obtained through tryptophan quenching experiments using brominated phospholipids and the recently developed lipid/polydiacetylene (PDA) colorimetric assay. The combined NMR, FTIR, fluorescence, and colorimetric studies shed light on the importance of segregation between the positive charges and the hydrophobic moieties on opposite surfaces within the peptides for facilitating membrane binding and disruption, compared to the formation of alpha helical or beta sheet structures.     

Synthesis and conformational studies of peptides encompassing the carboxy-terminal helix of thermolysin

The 21-residue fragment Tyr-Gly-Ser-Thr-Ser-Gln-Glu-Val-Ala-Ser-Val-Lys-Gln-Ala-Phe-Asp-Ala-Val- Gly-Val-Lys, corresponding to sequence 296-316 of thermolysin and thus encompassing the COOH-terminal helical segment 301-312 of the native protein, was synthesized by solid-phase methods and purified to homogeneity by reverse-phase high performance liquid chromatography. The peptide 296-316 was then cleaved with trypsin at Lys307 and Staphylococcus aureus V8 protease at Glu302, producing the additional fragments 296-307, 308-316, 296-302, and 303-316. All these peptides, when dissolved in aqueous solution at neutral pH, are essentially structureless, as determined by circular dichroism (CD) measurements in the far-ultraviolet region. On the other hand, fragment 296-316, as well as some of its proteolytic fragments, acquires significant helical conformation when dissolved in aqueous trifluoroethanol or ethanol. In general, the peptides mostly encompassing the helical segment 301-312 in the native thermolysin show helical conformation in aqueous alcohol. In particular, quantitative analysis of CD data indicated that fragment 296-316 attains in 90% aqueous trifluoroethanol the same percentage (approximately 58%) of helical secondary structure of the corresponding chain segment in native thermolysin. These results indicate that peptide 296-316 and its subfragments are unable to fold into a stable native-like structure in aqueous solution, in agreement with predicted location and stabilities of isolated subdomains of the COOH-terminal domain of thermolysin based on buried surface area calculations of the molecule.     

Alpha-agglutinin expression in Saccharomyces cerevisiae

A polyclonal antiserum raised against purified alpha-agglutinin was made specific for alpha-agglutinin after adsorption with a cells. The adsorbed antiserum identified alpha-agglutinin peptides on Western blots and bound to cell surface alpha-agglutinin, inhibiting the binding of alpha cells to a cells. Using the antibody, we have determined that 1) the surface distribution of alpha-agglutinin on alpha cells is polar, 2) about 5 x 10(4) molecules/cell are constitutively expressed on strain X2180-1B (alpha) cells, and 3) treatment of alpha cells with the sex pheromone a-factor causes an increase in cell surface alpha-agglutinin, consistent with the a-factor induced increase in cell agglutinability.     

Mapping of a cholinergic binding site by means of synthetic peptides, monoclonal antibodies, and alpha-bungarotoxin

Previous studies by several laboratories have identified a narrow sequence region of the nicotinic acetylcholine receptor (AChR) alpha subunit, flanking the cysteinyl residues at positions 192 and 193, as containing major elements of, if not all, the binding site for cholinergic ligands. In the present study, we used a panel of synthetic peptides as representative structural elements of the AChR to investigate whether additional segments of the AChR sequences are able to bind alpha-bungarotoxin (alpha-BTX) and several alpha-BTX-competitive monoclonal antibodies (mAbs). The mAbs used (WF6, WF5, and W2) were raised against native Torpedo AChR, specifically recognize the alpha subunit, and bind to AChR is inhibited by all cholinergic ligands. WF6 competes with agonists, but not with low mol. wt. antagonists, for AChR binding. The synthetic peptides used in this study were approximately 20 residue long, overlapped each other by 4-6 residues, and corresponded to the complete sequence of Torpedo AChR alpha subunit. Also, overlapping peptides, corresponding to the sequence segments of each Torpedo AChR subunit homologous to alpha 166-203, were synthesized. alpha-BTX bound to a peptide containing the sequence alpha 181-200 and also, albeit to a lesser extent, to a peptide containing the sequence alpha 55-74. WF6 bound to alpha 181-200 and to a lesser extent to alpha 55-74 and alpha 134-153. The two other mAbs predominantly bound to alpha 55-74, and to a lesser extent to alpha 181-200. Peptides alpha 181-200 and alpha 55-74 both inhibited binding of 125I-alpha-BTX to native Torpedo AChR. None of the peptides corresponding to sequence segments from other subunits bound alpha-BTX or WF6, or interfered with their binding. Therefore, the cholinergic binding site is not a single narrow sequence region, but rather two or more discontinuous sequence segments within the N-terminal extracellular region of the AChR alpha subunit, folded together in the native structure of the receptor, contribute to form a cholinergic binding region. Such a structural arrangement is similar to the "discontinuous epitopes" observed by X-ray diffraction studies of antibody-antigen complexes [reviewed in Davies et al. (1988)].     

Early events in the fibrillation of monomeric insulin

Insulin has a largely alpha-helical structure and exists as a mixture of hexameric, dimeric, and monomeric states in solution, depending on the conditions: the protein is monomeric in 20% acetic acid. Insulin forms amyloid-like fibrils under a variety of conditions, especially at low pH. In this study we investigated the fibrillation of monomeric human insulin by monitoring changes in CD, attenuated total reflectance-Fourier transform infrared spectroscopy, 8-anilinonaphthalenesulfonic acid fluorescence, thioflavin T fluorescence, dynamic light scattering, and H/D exchange during the initial stages of the fibrillation process to provide insight into early events involving the monomer. The results demonstrate the existence of structural changes occurring before the onset of fibril formation, which are detectable by multiple probes. The data indicate at least two major populations of oligomeric intermediates between the native monomer and fibrils. Both have significantly non-native conformations, and indicate that fibrillation occurs from a beta-rich structure significantly distinct from the native fold.     

Distinctive influence of two hexafluoro solvents on the structural stabilization of Bombyx mori silk fibroin protein and its derived peptides: 13C NMR and CD studies

Employing high-resolution (13)C solution NMR and circular dichroism (CD) spectroscopic techniques, the distinctive influence of two intimately related hexafluoro solvents, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and hexafluoroacetone trihydrate (HFA), on the structural characteristics of Bombyx mori (B. mori) silk fibroin, the chymotrypsin precipitate (C(p)) fraction, and two synthetic peptides, (AGSGAG)(5) and (AG)(15), is described. The observed (13)C solution NMR and CD spectra of these polypeptides in HFIP and HFA revealed a distinctive influence on their conformational characteristics. The (13)C NMR spectra, as analyzed from the unique chemical shifts of C(alpha) and C(beta) resonances of constituent residues revealed that fibroin largely assumes helical conformation(s) in both solvents. However, the peak shifts were greater for the samples in HFIP, indicating that the types of helical structure(s) may be different from the one populated in HFA. Similar structural tendencies of these polypeptides were reflected in CD spectra. The observed CD patterns, i.e., a strong positive band at approximately 190 nm and negative bands at approximately 206 and 222 nm, have been attributed to the preponderance of helical structures. Of the two prevalent helical structures, alpha-helix and 3(10)-helix, the evidence emerged for the fibroin protein in favor of 3(10)-helical structure stabilization in HFIP and its significant disruption in HFA, as deduced from the characteristic R1 (=[theta](190)/[theta](202)) and R2 (=[theta](222)/[theta](206)) ratios, determined from the CD data. Conversely, the native polypeptides and synthetic peptide fragments derived from highly crystalline regions of the silk fibroin protein sustained predominantly an unordered structure in HFA solvent.     

A conformational switch is associated with receptor affinity in peptides derived from the CD4-binding domain of gp120 from HIV I

A 15-residue region within the CD4-binding domain of gp120 from HIV I was identified with use of folding algorithms as conserving the potential for forming a particular secondary structure throughout 11 sequenced HIV strains. The region chosen has a potential for forming both beta-sheet and alpha-helix; the helical form would be amphipathic with the five hydrophobic residues all totally or functionally conserved. Five peptides were synthesized corresponding to this region in strain LAV and the strain most highly divergent from it in primary structure (Z3) plus three additional peptides with critical substitutions in the LAV sequence. The conformation of these five peptides was examined under various conditions with circular dichroism, and the results were compared with the ability of each peptide to bind to a CD4-expressing strain of HeLa cells (HeLa T4). In solution, the unmodified peptides exhibit a bistable structure, existing as beta-sheet in dilute buffer and converting to alpha-helix under more apolar conditions. The transition is reversible and sharp, occurring at a particular point in the polar/apolar gradient with virtually no intermediate state. The ability to undergo this bistable flip is closely associated with binding ability, amino acid substitutions that eliminate binding ability also eliminating the switch, and vice versa. The transition thus may reflect conformational changes occurring in this region of gp120 as it binds to the CD4 receptor.     

Conformational analysis of peptides corresponding to all the secondary structure elements of protein L B1 domain: secondary structure propensities are not conserved in proteins with the same fold.

The solution conformation of three peptides corresponding to the two beta-hairpins and the alpha-helix of the protein L B1 domain have been analyzed by circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR). In aqueous solution, the three peptides show low populations of native and non-native locally folded structures, but no well-defined hairpin or helix structures are formed. In 30% aqueous trifluoroethanol (TFE), the peptide corresponding to the alpha-helix adopts a high populated helical conformation three residues longer than in the protein. The hairpin peptides aggregate in TFE, and no significant conformational change occurs in the NMR observable fraction of molecules. These results indicate that the helical peptide has a significant intrinsic tendency to adopt its native structure and that the hairpin sequences seem to be selected as non-helical. This suggests that these sequences favor the structure finally attained in the protein, but the contribution of the local interactions alone is not enough to drive the formation of a detectable population of native secondary structures. This pattern of secondary structure tendencies is different to those observed in two structurally related proteins: ubiquitin and the protein G B1 domain. The only common feature is a certain propensity of the helical segments to form the native structure. These results indicate that for a protein to fold, there is no need for large native-like secondary structure propensities, although a minimum tendency to avoid non-native structures and to favor native ones could be required.     

Insights into the substrate specificity of plant peptide deformylase, an essential enzyme with potential for the development of novel biotechnology applications in agriculture

CD151, a member of the tetraspanin family of proteins, forms a stable complex with integrin alpha 3 beta 1 and regulates integrin-mediated cell-substrate adhesion. However, the molecular basis of the stable association of CD151 with integrin alpha 3 beta 1 remains poorly understood. In the present study, we show that a panel of anti-human CD151 mAbs (monoclonal antibodies) could be divided into three groups on the basis of their abilities to co-immunoprecipitate integrin alpha 3: Group-1 mAbs were devoid of sufficient activities to co-precipitate integrin alpha 3 under both low- and high-stringency detergent conditions; Group-2 mAbs co-precipitated integrin alpha 3 under low-stringency conditions; and Group-3 mAbs exhibited strong co-precipitating activities under both conditions. Group-1 mAbs in particular exhibited increased reactivity toward integrin alpha 3 beta 1-unbound CD151, indicating that the binding sites for Group-1 mAbs are partly blocked by bound integrin alpha 3 beta 1. Epitope mapping using a series of CD151 mutants with substitutions at amino acid residues that are not conserved between human and mouse CD151 revealed that Gly(176)/Gly(177), Leu(191) and Gln(194) comprise epitopes characteristic of Group-1 mAbs. Replacement of short peptide segments, each containing one of these epitopes, with those of other tetraspanins lacking stable interactions with integrin alpha 3 beta 1 demonstrated that the segment from Cys(185) to Cys(192), including Leu(191), was involved in the stable association of CD151 with integrin alpha 3 beta 1, as was the Gln(194)-containing QRD peptide. Taken together these results indicate that two consecutive segments including two Group-1 epitopes, Leu(191) and Gln(194), comprise an interface between CD151 and integrin alpha 3 beta 1, and, along with the epitope including Gly(176)/Gly(177), are concealed by bound integrin.     

Amyloid-forming peptides from beta2-microglobulin-Insights into the mechanism of fibril formation in vitro

Beta(2)-Microglobulin (beta(2)m) is one of over 20 proteins known to be involved in human amyloid disease. Peptides equivalent to each of the seven beta-strands of the native protein, together with an eighth peptide (corresponding to the most stable region in the amyloid precursor conformation formed at pH 3.6, that includes residues in the native strand E plus the eight succeeding residues (named peptide E')), were synthesised and their ability to form fibrils investigated. Surprisingly, only two sequences, both of which encompass the region that forms strand E in native beta(2)m, are capable of forming amyloid-like fibrils in vitro. These peptides correspond to residues 59-71 (peptide E) and 59-79 (peptide E') of intact beta(2)m. The peptides form fibrils under the acidic conditions shown previously to promote amyloid formation from the intact protein (pH <5 at low and high ionic strength), and also associate to form fibrils at neutral pH. Fibrils formed from these two peptides enhance fibrillogenesis of the intact protein. No correlation was found between secondary structure propensity, peptide length, pI or hydrophobicity and the ability of the peptides to associate into amyloid-like fibrils. However, the presence of a relatively high content of aromatic side-chains correlates with the ability of the peptides to form amyloid fibrils. On the basis of these results we propose that residues 59-71 may be important in the self-association of partially folded beta(2)m into amyloid fibrils and discuss the relevance of these results for the assembly mechanism of the intact protein in vitro.     

Stepwise proteolytic removal of the beta subdomain in alpha-lactalbumin. The protein remains folded and can form the molten globule in acid solution.

Bovine alpha-lactalbumin (alpha-LA) is an alpha/beta protein which adopts partly folded states when dissolved at low pH (A-state), by removal of the protein-bound calcium at neutral pH and low salt concentration (apo-state), as well as in aqueous trifluoroethanol. Previous spectroscopic studies have indicated that the A-state of alpha-LA at pH 2.0, considered a prototype molten globule, has a native-like fold in which the helical core is mostly retained, while the beta subdomain is less structured. Here, we investigate the conformational features of three derivatives of alpha-LA characterized by a single peptide bond fission or a deletion of 12 or 19/22 amino-acid residues of the beta subdomain of the native protein (approximately from residue 34 to 57). These alpha-LA derivatives were obtained by limited proteolysis of the protein in its partly folded state(s). A nicked alpha-LA species consisting of fragments 1-,3-40 and 41-123 (nicked-LA) was prepared by thermolytic digestion of the 123-residue chain of alpha-LA in 50% (v/v) aqueous trifluoroethanol. Two truncated or gapped protein species given by fragments 1-40 and 53-123 (desbeta1-LA) or fragments 1-34 and 54-,57-123 (desbeta2-LA) were obtained by digestion of alpha-LA with pepsin in acid or with proteinase K at neutral pH in its apo-state, respectively. The two protein fragments of nicked or gapped alpha-LA are covalently linked by the four disulfide bridges of the native protein. CD measurements revealed that, in aqueous solution at neutral pH and in the presence of calcium, the three protein species maintain the helical secondary structure of intact alpha-LA, while the tertiary structure is strongly affected by the proteolytic cleavages of the chain. Temperature effects of CD signals in the far- and near-UV region reveal a much more labile tertiary structure in the alpha-LA derivatives, while the secondary structure is mostly retained even upon heating. In acid solution at pH 2.0, the three alpha-LA variants adopt a conformational state essentially identical to the molten globule displayed by intact alpha-LA, as demonstrated by CD measurements. Moreover, they bind strongly the fluorescent dye 8-anilinonaphthalene-1-sulfonate, which is considered a diagnostic feature of the molten globule of proteins. Therefore, the beta subdomain can be removed from the alpha-LA molecule without impairing the capability of the rest of the chain to adopt a molten globule state. The results of this protein dissection study provide direct experimental evidence that in the alpha-LA molten globule only the alpha domain is structured.