Structure and properties of phospholipid-peptide monolayers containing monomeric SP-B(1-25) I. Phases and morphology by epifluorescence microscopy

Epifluorescence microscopy was used to study the structure and phase behavior of phospholipid films containing a human-sequence monomeric SP-B(1-25) synthetic peptide (mSP-B(1-25)). Measurements were done directly at the air-water (A/W) interface on films in a Langmuir-Whilhelmy balance coupled to a fluorescence microscope and real-time detection system to yield an approximate optical resolution of 1 mum. Fluorescence was achieved by laser excitation of 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-PC (BODIPY-PC, concentration 相似文献   

Effect of hydrophobic surfactant proteins SP-B and SP-C on binary phospholipid monolayers: II. Infrared external reflectance-absorption spectroscopy

In situ external reflection infrared spectroscopy at the air-water interface was used to study the influence on phospholipid structure of an endogenous mixture of the two hydrophobic surfactant proteins, SP-B and SP-C, which are thought to play pivotal roles in the adsorption and function of pulmonary surfactant. Mixtures studied were 1:1, 2:1, and 7:1 (mol:mol) DPPC-d(62):DPPG, and 7:1 DPPC-d(62):DOPG, alone and in the presence of 0.5-10 wt % mixed SP-B/C purified chromatographically from calf lung surfactant extract. Perdeuteration of DPPC produced a shift in vibrational frequencies so that it could be differentiated spectroscopically from the phosphoglycerol component in the surface monolayer. CH(2) antisymmetric and symmetric stretching bands ( approximately 2920 and 2852 cm(-1)) along with the analogous CD(2) stretching bands ( approximately 2194 and 2089 cm(-1)) were analyzed, and band heights and peak wavenumber positions were assessed as a function of monolayer surface pressure. Small, near-physiological contents of 1-2 wt % SP-B/C typically produced the maximum observed spectroscopic effects, which were abolished at high protein contents of 10 wt %. Analysis of CH(2) and CD(2) stretching bands and C-H/C-D band height ratios indicated that SP-B/C affected PC and PG lipids differently within the surface monolayer. SP-B/C had preferential interactions with DPPG in 1:1, 2:1, and 7:1 DPPC-d(62):DPPG films that increased its acyl chain order. SP-B/C also interacted specifically with DOPG in 7:1 DPPC-d(62):DOPG monolayers, but in this case an increase in CH(2) band heights and peak wavenumber positions indicated a further disordering of the already fluid DOPG acyl chains. CD(2) band height and peak wavenumber analysis indicated that SP-B/C had no significant effect on the structure of DPPC-d(62) chains in 7:1 films with DPPG or DOPG, and had only a slight tendency to increase the acyl chain order in 1:1 films of DPPC-d(62):DPPG. SP-B/C had no significant effect on DPPC-d(62) structure in films with DOPG. Infrared results also indicated that interactions involving SP-B/C and lipids led to exclusion of PC and PG lipids from the compressed interfacial monolayer, in agreement with our previous report on the phase morphology of lipid monolayers containing 1 wt % SP-B/C.     

Surface active properties of amphiphilic sequential isopeptides: Comparison between alpha-helical and beta-sheet conformations

Poly(Leu-Lys-Lys-Leu) and poly(Leu-Lys) are sequential amphiphilic peptide isomers that adopt respectively an alpha-helical conformation and a beta-sheet structure in saline solutions and at the air/water interface. The surface active properties of LKKL and LK sequential isopeptides containing 16, 20, and n residues have been compared in order to evaluate the contributions of the alpha-helical and beta-sheet conformations. Both have a natural tendency to spread at the surface of a saline solution and the values of the equilibrium spreading pressure pi(e) lie in the same range. When dissolved in a saline solution, alpha-helical peptides diffuse faster and adsorb faster at the interface than the beta-sheet isomers. From the compression isotherms of LKKL and LK peptide monolayers it is possible to extract parameters that characterize the behavior of alpha-helical and beta-sheet conformations: beta-sheet peptide monolayers are more stable and less compressible than the monolayers formed with the alpha-helical isomers. The LK peptides differ also by their high degree of self-association at the air/water interface. Copyright 1999 John Wiley & Sons, Inc.     

Effect of hydrophobic surfactant proteins SP-B and SP-C on phospholipid monolayers. Protein structure studied using 2D IR and beta correlation analysis

We have applied two-dimensional infrared (2D IR) and betanu correlation spectroscopy to in-situ IR spectroscopy of pulmonary surfactant proteins SP-B and SP-C in lipid-protein monolayers at the air-water interface. For both SP-B and SP-C, a statistical windowed autocorrelation method identified two separate surface pressure regions that contained maximum amide I intensity changes: 4-25 mN/m and 25-40 mN/m. For SP-C, 2D IR and betanu correlation analyses of these regions indicated that SP-C adopts a variety of secondary structure conformations, including alpha-helix, beta-sheet, and an intermolecular aggregation of extended beta-sheet structure. The main alpha-helix band split into two peaks at high surface pressures, indicative of two different helix conformations. At low surface pressures, all conformations of the SP-C molecule reacted identically to increasing surface pressure and reoriented in phase with each other. Above 25 mN/m, however, the increasing surface pressure selectively affected the coexisting protein conformations, leading to an independent reorientation of the protein conformations. The asynchronous 2D IR spectrum of SP-B showed the presence of two alpha-helix components, consistent with two separate populations of alpha-helix in SP-B-a hydrophobic fraction associated with the lipid chains and a hydrophilic fraction parallel to the membrane surface. The distribution of correlation intensity between the two alpha-helix cross peaks indicated that the more hydrophobic helix fraction predominates at low surface pressures whereas the more hydrophilic helix fraction predominates at high surface pressures. The different SP-B secondary structures reacted identically to increasing surface pressure, leading to a reorientation of all SP-B subunits in phase with one another.     

The conformation of glucagon: predictions and consequences.

It is proposed that glucagon, a polypeptide hormone, is delicately balanced between two major conformational states. Utilizing a new predictive model [Chou, P.Y., and Fasman, G.D. (1974), Biochemistry 13, 222] which considers all the conformational states in proteins (helix, beta sheet, random coil, and beta turns), the secondary structural regions of glucagon are computed herein. The conformational sensitivity of glucagon may be due to residues 19-27 which have both alpha-helical potential (mean value of Palpha = 1.19) as well as beta-sheet potential (mean value of Pbeta = 1.25). Two conformational states are predicted for glucagon. In predicted form (a), residues 5-10 form a beta-sheet region while residues 19-27 form an alpha-helical region (31% alpha, 21% beta) agreeing well with the circular dichroism (CD) spectra of glucagon. The similarity in the CD spectra of glucagon and insulin further suggests the presence of beta structure in glucagon, since X-ray analysis of insulin showed 24% beta sheet. In predicted form (b), both regions, residues 5-10 and residues 19-27, are beta sheets sheets (0% alpha, 52% beta) in agreement with the infrared spectral evidence that glucagon gels and fibrils have a predominant beta-sheet conformation. Since three reverse beta turns are predicted at residues 2-5, 10-13, and 15-18, glucagon may possess tertiary structure in agreement with viscosity and tritium-hydrogen exchange experiments. A proposal is offered concerning an induced alpha yields beta transition at residues 22-27 in glucagon during receptor site binding. Amino acid substitutions are proposed which should disrupt the beta sheets of glucagon with concomitant loss of biological activity. The experimental findings that glucagon aggregates to form dimers, trimers, and hexamers can be explained in terms of beta-sheet interactions as outlined in the present predictive model. Thus the conflicting conclusions of previous workers, concerning the conformation of glucagon in different environments, can be rationalized by the suggested conformational transition occurring within the molecule.     

Solution conformations and aggregational properties of synthetic amyloid beta-peptides of Alzheimer's disease. Analysis of circular dichroism spectra.

The A4 or beta-peptide (39 to 43 amino acid residues) is the principal proteinaceous component of amyloid deposits in Alzheimer's disease. Using circular dichroism (c.d.), we have studied the secondary structures and aggregational properties in solution of 4 synthetic amyloid beta-peptides: beta-(1-28), beta-(1-39), beta-(1-42) and beta-(29-42). The natural components of cerebrovascular deposits and extracellular amyloid plaques are beta-(1-39) and beta-(1-42), while beta-(1-28) and beta-(29-42) are unnatural fragments. The beta-(1-28), beta-(1-39) and beta-(1-42) peptides adopt mixtures of beta-sheet, alpha-helix and random coil structures, with the relative proportions of each secondary structure being strongly dependent upon the solution conditions. In aqueous solution, beta-sheet structure is favored for the beta-(1-39) and beta-(1-42) peptides, while in aqueous solution containing trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP), alpha-helical structure is favored for all 3 peptides. The alpha-helical structure unfolds with increasing temperature and is favored at pH 1 to 4 and pH 7 to 10; the beta-sheet conformation is temperature insensitive and is favored at pH 4 to 7. Peptide concentration studies showed that the beta-sheet conformation is oligomeric (intermolecular), whereas the alpha-helical conformation is monomeric (intramolecular). The rate of aggregation to the oligomeric beta-sheet structure (alpha-helix----random coil----beta-sheet) is also dependent upon the solution conditions such as the pH and peptide concentration; maximum beta-sheet formation occurs at pH 5.4. These results suggest that beta-peptide is not an intrinsically insoluble peptide. Thus, solution abnormalities, together with localized high peptide concentrations, which may occur in Alzheimer's disease, may contribute to the formation of amyloid plaques. The hydrophobic beta-(29-42) peptide adopts exclusively an intermolecular beta-sheet conformation in aqueous solution despite changes in temperature or pH. Therefore, this segment may be the first region of the beta-peptide to aggregate and may direct the folding of the complete beta-peptide to produce the beta-pleated sheet structure found in amyloid deposits. Differences between the solution conformations of the beta-(1-39) and beta-(1-42) peptides suggests that the last 3 C-terminal amino acids are crucial to amyloid deposition.     

Structural transitions involved in a novel amyloid-like beta-sheet assemblage of tripeptide derivatives

Self-assembly of two tripeptide derivatives containing three nonpolar isoleucine moieties and polar oxyethylene groups are studied in methanol. Peptide A [CH3(OCH2CH2)3OCH2CO(Ile)3OCH3] and peptide B [CH3(OCH2CH2)3OCH2CO(Ile)3NH (CH2CH2O)3CH3] take a mixture of unordered and helical conformation at low concentration (8.5 x 10(-4) M). However, at high concentration (2 x 10(-3) M), both the peptide showed significant increase in the helical conformation. An interesting conformational transition of peptides A and B at various methanol contents was observed in the solvated films of these compounds by spectroscopic methods like the far-uv circular dichroism and Fourier transform infrared (FT-IR) techniques. Peptide B, which contains more polar oxyethylene groups than A, showed a highly cooperative conformational transition when the methanol content was decreased. This transition was characterized by a large increase of beta-sheet, retaining a alpha-helical contribution. Peptide A showed a conformational transition resulting in a beta-sheet in the aggregated state. From the CD spectra, the ratio in the ellipticity indicates that peptide B forms twisted antiparallel beta-sheet conformation, whereas peptide A takes a parallel beta-sheet conformation. The results obtained in this work indicates the role of polar derivatization on the conformational preference of peptides having similar sequence.     

Role of water molecules in the crystal structure of gly-L-ala-L-phe: A possible sequence preference for nucleation of α-helix?

The synthetic peptide Gly-L-Ala-L-Phe (C14H19N3O4.2H2O; GAF) crystallizes in the monoclinic space group P2I1), with a = 5.879(1), b = 7.966(1), c = 17.754(2) A, beta = 95.14(2) degrees, Dx = 1.321 g cm-3, and Z = 2. The crystal structure was solved by direct methods using the program SHELXS-86 and refined to an R value of 0.031 for 1425 reflections (greater than 3 sigma). The tripeptide exists as a zwitterion in the crystal and assumes a near alpha-helical backbone conformation with the following torsion angles: psi 1 = -147.8 degrees; phi 2, psi 2 = -71.2 degrees, 33.4 degrees; phi 3, psi 3 = -78.3 degrees, -43.3 degrees. In this structure, one water molecule bridges the COO- and NH3+ terminii to complete a turn of an alpha-helix and another water molecule participates in head-to-tail intermolecular hydrogen bonding, so that the end result is a column of molecules that looks like an alpha-helix. Thus, the two water molecules of crystallization play a major role in stabilizing the near alpha-helical conformation of each tripeptide molecule and in elongating the helix throughout the crystal. An analysis of all protein sequences around regions containing a GAF fragment by Chou-Fasman's secondary structure prediction method showed that those regions are likely to assume an alpha-helical conformation with twice the probability they are likely to adopt a beta-sheet conformation. It is conceivable that a GAF fragment may be a good part of the nucleation site for forming alpha-helical fragments in a polypeptide, with the aqueous medium playing a crucial role in maintaining such transient species.     

Circular dichroism and Fourier transform infrared spectroscopic studies on self-assembly of tetrapeptide derivative in solution and solvated film.

Aggregation of the hydrophobic peptide derivative Boc-Ala-Ile-Ile-Gly-OMe (1) was examined in methanol solution and in solvated film states. Formation of the peptide by self-assembly was evidenced using fluorescence [Mg salt of 8-anilino-naphthalenesulfonic acid (ANS) as an external probe] and circular dichroism (CD) spectroscopic techniques. In solution, peptide 1 formed as a stable aggregate at a concentration around 3 x 10(-4)m. The peptide gelled into a thin film for which we carried out CD and Fourier transform infrared (FTIR) measurements. Our spectroscopic study on peptide films at differing methanol concentrations indicates that the helical content of the peptide decreases with decreasing methanol concentration in solvated films. However, by reducing the methanol concentration we were able to observe a conformational transition from a predominantly helical turn to a beta-sheet structure via a random coil conformation. Our study focused on the aggregation of the alpha-helical turn-forming peptide derivative, which shows conformational transition on changing solvent concentration in the film form.     

Conformational mapping of the N-terminal segment of surfactant protein B in lipid using 13C-enhanced Fourier transform infrared spectroscopy.

Synthetic peptides based on the N-terminal domain of human surfactant protein B (SP-B1-25; 25 amino acid residues; NH2-FPIPLPYCWLCRALIKRIQAMIPKG) retain important lung activities of the full-length, 79-residue protein. Here, we used physical techniques to examine the secondary conformation of SP-B1-25 in aqueous, lipid and structure-promoting environments. Circular dichroism and conventional, 12C-Fourier transform infrared (FTIR) spectroscopy each indicated a predominate alpha-helical conformation for SP-B1-25 in phosphate-buffered saline, liposomes of 1-palmitoyl-2-oleoyl phosphatidylglycerol and the structure-promoting solvent hexafluoroisopropanol; FTIR spectra also showed significant beta- and random conformations for peptide in these three environments. In further experiments designed to map secondary structure to specific residues, isotope-enhanced FTIR spectroscopy was performed with 1-palmitoyl-2-oleoyl phosphatidylglycerol liposomes and a suite of SP-B1-25 peptides labeled with 13C-carbonyl groups at either single or multiple sites. Combining these 13C-enhanced FTIR results with energy minimizations and molecular simulations indicated the following model for SP-B1-25 in 1-palmitoyl-2-oleoyl phosphatidylglycerol: beta-sheet (residues 1-6), alpha-helix (residues 8-22) and random (residues 23-25) conformations. Analogous structural motifs are observed in the corresponding homologous N-terminal regions of several proteins that also share the 'saposin-like' (i.e. 5-helix bundle) folding pattern of full-length, human SP-B. In future studies, 13C-enhanced FTIR spectroscopy and energy minimizations may be of general use in defining backbone conformations at amino acid resolution, particularly for peptides or proteins in membrane environments.     

Comparative interaction of alpha-helical and beta-sheet amphiphilic isopeptides with phospholipid monolayers

The two sequential amphiphilic peptide isomers, (Leu-Lys-Lys-Leu)4 and (Leu-Lys)8, were chosen as models for alpha-helical and beta-sheet peptides, respectively. In order to evaluate the contribution of the secondary structure of a peptide to its penetration into cellular membranes, interactions of these isopeptides with L-alpha-dimyristoyl phosphatidylcholine (DMPC) monolayers were studied. Both isopeptides penetrate into DMPC monolayers up to an exclusion pressure of approximately 27 mN/m, but a discontinuity is observed in the penetration profile of the alpha-helical (LKKL)4. The main parameters extracted from the compression isotherms of the mixed peptide/DMPC monolayers-namely, transition pressure, mean area, elasticity modulus, and energy of mixing-were analyzed. These analyses indicate that the alpha-helical isomer interacts strongly with DMPC by forming a 1:32 (LKKL)4-DMPC complex. When engaged in this complex, (LKKL)(4) behaves as an hydrophobic helix and has a tendency to become vertically oriented in the course of the compression process. The beta-sheet (LK)8 interacts more weakly with DMPC and no complex can be detected.     

Product conformation driven splicing of unprotected peptides by reverse proteolysis: influence of intrinsic and extrinsic factors

The structural motif of 'product conformation driven V8 protease catalyzed ligation reaction' can be represented by FR(I)-EALER-FR(II). The relative roles of the flanking regions (FR(I) and FR(II)) and of splicedon, the central penta-peptide, on the thermodynamic stability of the 'conformational trap' of the product has been now evaluated as a function of co-solvent concentration. The studies have established that the thermodynamic stability of the conformational trap of alpha17-40des23-26 with four different splicedons (EALER, EALEV, EYGER, or EGAER) that differ in the intrinsic alpha-helical potential of their amino acid residues and/or ability to generate i, i+4 side chain interaction is a direct correlate of the n-propanol induced alpha-helical conformation of the product. On the other hand, when the product is defined by only splicedon EALER, and the flanking regions are disitinct; no correlation could be drawn between the stability of the trap and solvent induced alpha-helical conformation, even though these generally give an equilibrium yield of 45% in 30% n-propanol and is not influenced by an increased propanol concentration. However, when the splicedon EALER with given FR(I) and FR(II) region develops a 'conformational trap' of a lower stability in 30% propanol as seen with beta18-25(A22)-EALER-beta31-39, the stability increases in 60% n-propanol, without significant increase in the alpha- helical conformation. Though, primary structure of RNAse1-20, could be presented as RNAse1-5-AKFER- RNAse1-20, and alpha-helical conformation is induced to this peptide both in 30 and 60% propanol, splicedon AKFER by itself does not develop the 'conformational trap' of RNAse1-20. The splicedon AKFER of RNAse1-20 fails to develop the 'conformational trap', due to an intrinsic inhibitory potential of its FR region, RNAse11-20; replacing RNAse11-20 with alpha32-40 enables the splicedon AFKER to generate the 'conformational trap'. The studies presented here have demonstrated the primary role of flanking regions in establishing the amount of the solvent induced alpha-helical conformation and that of the splicedon in dictating the thermodynamic stability of its 'conformational trap' of the products, nonetheless one influences the other to some degree. We suggest that the stability of the 'conformational trap' of the product reflects the ability of the splicedon to 'recruit' the product conformation to protect the spliced peptide bond, i.e. to reduce the helix-coil transition of the spliced region which in turn imparts a degree of resistance to the spliced peptide bond.     

Interaction of cationic antimicrobial peptides with model membranes

A series of natural and synthetic cationic antimicrobial peptides from various structural classes, including alpha-helical, beta-sheet, extended, and cyclic, were examined for their ability to interact with model membranes, assessing penetration of phospholipid monolayers and induction of lipid flip-flop, membrane leakiness, and peptide translocation across the bilayer of large unilamellar liposomes, at a range of peptide/lipid ratios. All peptides were able to penetrate into monolayers made with negatively charged phospholipids, but only two interacted weakly with neutral lipids. Peptide-mediated lipid flip-flop generally occurred at peptide concentrations that were 3- to 5-fold lower than those causing leakage of calcein across the membrane, regardless of peptide structure. With the exception of two alpha-helical peptides V681(n) and V25(p,) the extent of peptide-induced calcein release from large unilamellar liposomes was generally low at peptide/lipid molar ratios below 1:50. Peptide translocation across bilayers was found to be higher for the beta-sheet peptide polyphemusin, intermediate for alpha-helical peptides, and low for extended peptides. Overall, whereas all studied cationic antimicrobial peptides interacted with membranes, they were quite heterogeneous in their impact on these membranes.     

The intact human acetylcholinesterase C-terminal oligomerization domain is alpha-helical in situ and in isolation,but a shorter fragment forms beta-sheet-rich amyloid fibrils and protofibrillar oligomers

A 14-residue fragment of the C-terminal oligomerization domain, or T-peptide, of human acetylcholinesterase (AChE) shares sequence homology with the amyloid-beta peptide implicated in Alzheimer's disease and can spontaneously self-assemble into classical amyloid fibrils under physiological conditions [Greenfield, S. A., and Vaux, D. J. (2002) Neuroscience 113, 485-492; Cottingham, M. G., Hollinshead, M. S., and Vaux, D. J. (2002) Biochemistry 41, 13539-13547]. Here we demonstrate that the conformation of this AChE(586-599) peptide, both before and after fibril formation, is different from that of a longer peptide, T(40), corresponding to the entire 40-amino acid T-peptide (residues 575-614 of AChE). This peptide is prone to homomeric hydrophobic interactions, consistent with its role in AChE subunit assembly, and possesses an alpha-helical structure which protects against the development of the beta-sheet-rich amyloidogenic conformation favored by the shorter constituent AChE(586-599) fragment. Using a conformation-sensitive monoclonal antibody raised against the alpha-helical T(40) peptide, we demonstrate that the conformation of the T-peptide domain within intact AChE is antigenically indistinguishable from that of the synthetic T(40) peptide. A second monoclonal antibody raised against the fibrillogenic AChE(586-599) fragment recognizes not only beta-sheet amyloid aggregates but also SDS-resistant protofibrillar oligomers. A single-antibody sandwich ELISA confirms that such oligomers exist at micromolar peptide concentrations, well below that required for formation of classical amyloid fibrils. Epitope mapping with this monoclonal antibody identifies a region near the N-terminus of the peptide that remains accessible in oligomer and fibril alike, suggesting a model for the arrangement of subunits within AChE(586-599) protofibrils and fibrils.     

Reconstitution of the M13 major coat protein and its transmembrane peptide segment on a DNA template

The major coat protein (pVIII) of M13 phage is of particular interest to structure biologists since it functions in two different environments: during assembly and infection, it interacts with the bacterial lipid bilayer, but in the phage particle, it exists as a protein capsid to protect a closed circular, single-stranded DNA (ssDNA) genome. We synthesized pVIII and a 32mer peptide consisting of the transmembrane and DNA binding domains of pVIII. The 32mer peptide displays typically an alpha-helical structure in trifluroethanol or 0.2 M octylglucoside solutions similar to pVIII. Attachment of polyethylene glycol (PEG) onto the N-terminal of 32mer increased the alpha-helical content and the peptide thermal stability. The peptides were reconstituted with DNA from a detergent solution into a discrete (<200 nm diameter) nanoparticle on both linear double-stranded DNA (dsDNA) and linear ssDNA, where the linear dsDNA is used to mimic the closed circular, ssDNA in M13 phage, upon removal of the detergent. The peptide/DNA particle was an irregular and not a rod-shaped aggregate when imaged by atomic force microscopy. All three peptides underwent a structural transition from alpha-helix to beta-sheet within approximately 1 h of DNA addition to the detergent solution. There was a further decrease in alpha-helical content when the detergent was removed. The presence of anionic (such as octanoic acid) or cationic (such as 1,5-diaminopentane) molecules in the detergent mixture resulted in the retention of the peptide alpha-helical structure. Thus the interaction between the peptide and DNA in octylglucoside is driven by electrostatic forces, and peptide-peptide interactions are responsible for the transition from alpha-helix to beta-sheet conformation in pVIII and its analogues. These results suggest that the assembly process to form a rod-shaped phage is a delicate balance to maintain pVIII in an alpha-helical conformation that requires either an oriented bilayer to solubilize pVIII prior to interaction with the DNA or other phage proteins to nucleate pVIII in the alpha-helical conformation on the DNA.     

NMR studies of amyloid beta-peptides: proton assignments, secondary structure, and mechanism of an alpha-helix----beta-sheet conversion for a homologous, 28-residue, N-terminal fragment.

Beta-peptide is a major component of amyloid deposits in Alzheimer's disease. We report here a proton nuclear magnetic resonance (NMR) spectroscopic investigation of a synthetic peptide that is homologous to residues 1-28 of beta-peptide [abbreviated as beta-(1-28)]. The beta-(1-28) peptide produces insoluble beta-pleated sheet structures in vitro, similar to the beta-pleated sheet structures of beta-peptide in amyloid deposits in vivo. For peptide solutions in the millimolar range, in aqueous solution at pH 1-4 the beta-(1-28) peptide adopts a monomeric random coil structure, and at pH 4-7 the peptide rapidly precipitates from solution as an oligomeric beta-sheet structure, analogous to amyloid deposition in vivo. The NMR work shown here demonstrates that the beta-(1-28) peptide can adopt a monomeric alpha-helical conformation in aqueous trifluoroethanol solution at pH 1-4. Assignment of the complete proton NMR spectrum and the determination of the secondary structure were arrived at from interpretation of two-dimensional (2D) NMR data, primarily (1) nuclear Overhauser enhancement (NOE), (2) vicinal coupling constants between the amide (NH) and alpha H protons, and (3) temperature coefficients of the NH chemical shifts. The results show that at pH 1.0 and 10 degrees C the beta-(1-28) peptide adopts an alpha-helical structure that spans the entire primary sequence. With increasing temperature and pH, the alpha-helix unfolds to produce two alpha-helical segments from Ala2 to Asp7 and Tyr10 to Asn27. Further increases in temperature to 35 degrees C cause the Ala2-Asp7 section to become random coil, while the His13-Phe20 section stays alpha-helical. A mechanism involving unfavorable interactions between charged groups and the alpha-helix macrodipole is proposed for the alpha-helix----beta-sheet conversion observed at midrange pH.     

Conformational polymorphism of the amyloidogenic peptide homologous to residues 113-127 of the prion protein

Conformational transitions are thought to be the prime mechanism of amyloid formation in prion diseases. The prion proteins are known to exhibit polymorphic behavior that explains their ability of "conformation switching" facilitated by structured "seeds" consisting of transformed proteins. Oligopeptides containing prion sequences showing the polymorphism are not known even though amyloid formation is observed in these fragments. In this work, we have observed polymorphism in a 15-residue peptide PrP (113-127) that is known to form amyloid fibrils on aging. To see the polymorphic behavior of this peptide in different solvent environments, circular dichroism (CD) spectroscopic studies on an aqueous solution of PrP (113-127) in different trifluoroethanol (TFE) concentrations were carried out. The results show that PrP (113-127) have sheet preference in lower TFE concentration whereas it has more helical conformation in higher TFE content (>40%). The structural transitions involved in TFE solvent were studied using interval-scan CD and FT-IR studies. It is interesting to note that the alpha-helical structure persists throughout the structural transition process involved in amyloid fibril formation implicating the involvement of both N- and C-terminal sequences. To unravel the role of the N-terminal region in the polymorphism of the PrP (113-127), CD studies on another synthetic peptide, PrP (113-120) were carried out. PrP(113-120) exhibits random coil conformation in 100% water and helical conformation in 100% TFE, indicating the importance of full-length sequence for beta-sheet formation. Besides, the influence of different chemico-physical conditions such as concentration, pH, ionic strength, and membrane like environment on the secondary structure of the peptide PrP (113-127) has been investigated. At higher concentration, PrP (113-127) shows features of sheet conformation even in 100% TFE suggesting aggregation. In the presence of 5% solution of sodium dodecyl sulfate, PrP (113-127) takes high alpha-helical propensity. The environment-dependent conformational polymorphism of PrP (113-127) and its marked tendency to form stable beta-sheet structure at acidic pH could account for its conformation switching behavior from alpha-helix to beta-sheet. This work emphasizes the coordinative involvement of N-terminal and C-terminal sequences in the self-assembly of PrP (113-127).     

Solution stability of salmon calcitonin at high concentration for delivery in an implantable system.

Salmon calcitonin solutions (50 mg/mL and 100 mg/mL) were placed on stability at 37 degrees C for 1 year in a variety of solvent systems including water, ethanol, glycerol, propylene glycol (PG) and dimethyl sulfoxide (DMSO). Calcitonin degradation was monitored by RP-HPLC and size-exclusion chromatography. DMSO and pH 3.3 solutions provided optimum stability. Conformational stability was also monitored by FTIR over the 1 year time course and compared with chemical and physical stability. After 12 months at 37 degrees C, four major conformations were observed: a beta-sheet conformation (pH 3.3, pH 5.0, 70% DMSO and 70% glycerol), an aggregate conformation (pH 7.0 water), a strong alpha-helical conformation (70% EtOH, 70% PG) and a weak alpha-helical conformation (100% DMSO). No correlation between structure and chemical stability was observed in which both the beta-sheet structure (pH 3.3, water) and a loose alpha-helical structure (100% DMSO) demonstrated good stability. However, some correlation was observed between structure and physical stability, where co-solvents inducing an alpha-helical structure resulted in a decrease in gelation. These two structural states associated with improved stability and minimal gelation, indicated that gelation can be reduced or eliminated by the use of pharmaceutically acceptable co-solvents. Finally, salmon calcitonin (50 mg/mL) was formulated in 100% DMSO and delivered from a DUROS implant over 4 months. Delivery at a target dose of 18 microg/day calcitonin at 37 degrees C was confirmed.     

Product-conformation-driven ligation of peptides by V8 protease

Organic co-solvent-induced secondary conformation of alpha(17-40) of human hemoglobin facilitates the splicing of E30-R31 in a mixture of its complementary segments by V8 protease. The amino acid sequence of alpha(17-40) has been conceptualized by the general structure FR(I)-EALER-FR(II) and the pentapeptide sequence EALER playing a major role in inducing the alpha-helical conformation. The primary structure of alpha(17-40) has been engineered in multiple ways to perturb one, two, or all three regions and the influence of the organic co-solvent-induced conformation and the concomitant resistance of E30-R31 peptide bond to V8 protease digestion has been investigated. The central pentapeptide (EALER), referred to here as splicedon,(3) appears to dictate a primary role in facilitating the splicing reaction. When the same flanking regions are used, (1) splicedons that carry amino acid residues of low alpha-helical potential, for example G at position 2 or 3 of the splicedon, generate a conformational trap of very low thermodynamic stability, giving an equilibrium yield of only 3%-5%; (2) splicedons with amino acid residues of good alpha-helical potential generate a conformational trap of medium thermodynamic stability and give an equilibrium yield of 20%-25%; (3) the splicedons with amino residues of good alpha-helical potential and also an amino acid that can generate an i, i + 4 side-chain carboxylate-guanidino (amino) interaction, a conformational trap of maximum thermodynamic stability is generated, giving an equilibrium yield of 45%-50%; and (4) the thermodynamic stability of the conformational trap of the spliced peptide is also influenced by the amino acid composition of the flanking regions. The V8 protease resistance of the spliced peptide bond is not a direct correlate of the amount of alpha-helical conformation induced into the product. The results of this study reflect the unique role of the splicedon in translating the organic co-solvent-induced product conformation as a site-specific stabilization of the spliced peptide bond. It is speculated that the splicedon with higher alpha-helical potential as compared to either one of the flanking regions achieves this by integrating its potential with that of the flanking region(s). Exchange of flanking regions with the products of other V8 protease-catalyzed splicing reactions will help to establish the general primary structural requirements of this class of splicing reactions and facilitate their application in modular construction of proteins.     

Further investigation on potassium-induced conformation transition of Nephila spidroin film with two-dimensional infrared correlation spectroscopy

We used two-dimensional (2D) correlation infrared spectroscopy to study further the potassium-induced conformation transition in Nephila spidroin films. It provided increased resolution and important new information on the sequence of events in the conformation transition process, showing that beta-sheet formed from the helical component before they formed from random coil. It also showed more evidence that formation of the 1691 cm(-1) (turn/bend) peak did not proceed with the same kinetics as the 1620 cm(-1) (antiparallel beta-sheet component) one, so we attribute the 1691 cm(-1) peak to turns which formed with different kinetics as the antiparallel beta-sheets. We present a single coherent and detailed hypothesis for the assembly and secondary structural transition of silk proteins in vivo and in vitro based on our findings and on evidence from other laboratories.