The relation of predicted structure to observed conformation and activity of glucagon analogs containing replacements at positions 19, 22, and 23

Six new analogs of glucagon have been synthesized containing replacements at positions 19, 22, and 23. They were designed to study the correlation between predicted conformation in the 19-27 segment of the hormone and the conformation calculated from circular dichroism measurements and the observed activation of adenylate cyclase in the liver membrane. The analogs were [Val19]glucagon, [Val22]glucagon, [Glu23]glucagon, [Val19,Glu23]glucagon, [Glu22,Glu23]glucagon, and [Ala22,Ala23]glucagon. The structures predicted for the 19-27 segment ranged from strongly alpha helical to weakly beta sheet. The observed conformations varied as functions of amino acid composition, solvent, concentration, pH, and temperature but did not correlate well with prediction. There was, however, a correlation between predicted structure and activation of adenylate cyclase in rat liver membranes.     

Fourier transform infared spectroscopy investigation of protein conformation in spray-dried protein/trehalose powders

Spray drying is a way to generate protein solids (powders), which is also true for lyophilization. Sugars are used to protect proteins from conformational changes and chemical degradations arising from drying processes and storage conditions such as the humidity. The influence of trehalose and humidity on the conformation and hydration of spray-dried recombinant human granolucyte colony stimulating factor (rhG-CSF) and recombinant consensus interferon-alpha (rConIFN) was investigated using Fourier transform IR spectroscopy. The spectral analysis of spray-dried powders in the amide I region demonstrated that trehalose stabilized the alpha-helical conformation of both rhG-CSF and rConIFN proteins. Exposure of the pure protein powders to 33% relative humidity (RH) resulted in the formation of beta sheets and loss of turns but no change in alpha-helical structure. Trehalose reduced the magnitude of the changes in beta sheets and turns. Exposure of the pure protein powders to 75% RH resulted in the loss of alpha-helical conformation with a corresponding increase in beta structures (beta sheets and turns). Trehalose did not protect proteins from the loss of alpha-helical structures, but it reduced the formation of antiparallel beta sheets. Hydrogen-deuterium exchange (H-D exchange) was used to further characterize these hydration-induced conformational changes. At 33% RH the percent exchange of the protein decreased with increasing trehalose content, indicating a greater protection of the protein from H-D exchange by a higher concentration of trehalose. Such protection correlates with decreased conformational changes of the protein by trehalose at this humidity. At 75% RH the degree of H-D exchange of the protein was insensitive to the powder composition in all powders. Surprisingly, the H-D exchange of trehalose was low at about 20-25%, which was nearly independent of the protein/trehalose ratio and humidity, indicating that the exchangeable protons on trehalose molecules are highly protected in protein-containing powders. The observed protein hydration is related to the effect of trehalose on the conformational changes of the protein under humidity.     

Solid-state NMR studies of the prion protein H1 fragment.

Conformational changes in the prion protein (PrP) seem to be responsible for prion diseases. We have used conformation-dependent chemical-shift measurements and rotational-resonance distance measurements to analyze the conformation of solid-state peptides lacking long-range order, corresponding to a region of PrP designated H1. This region is predicted to undergo a transformation of secondary structure in generating the infectious form of the protein. Solid-state NMR spectra of specifically 13C-enriched samples of H1, residues 109-122 (MKHMAGAAAAGAVV) of Syrian hamster PrP, have been acquired under cross-polarization and magic-angle spinning conditions. Samples lyophilized from 50% acetonitrile/50% water show chemical shifts characteristic of a beta-sheet conformation in the region corresponding to residues 112-121, whereas samples lyophilized from hexafluoroisopropanol display shifts indicative of alpha-helical secondary structure in the region corresponding to residues 113-117. Complete conversion to the helical conformation was not observed and conversion from alpha-helix back to beta-sheet, as inferred from the solid-state NMR spectra, occurred when samples were exposed to water. Rotational-resonance experiments were performed on seven doubly 13C-labeled H1 samples dried from water. Measured distances suggest that the peptide is in an extended, possibly beta-strand, conformation. These results are consistent with the experimental observation that PrP can exist in different conformational states and with structural predictions based on biological data and theoretical modeling that suggest that H1 may play a key role in the conformational transition involved in the development of prion diseases.     

Chain-length dependence of alpha-helix to beta-sheet transition in polylysine: model of protein aggregation studied by temperature-tuned FTIR spectroscopy

The chain-length dependence of the alpha-helix to beta-sheet transition in poly(L-lysine) is studied by temperature-tuned FTIR spectroscopy. This study shows that heterogeneous samples of poly(L-lysine), comprising polypeptide chains with various lengths, undergo the alpha-beta transition at an intermediate temperature compared to homogeneous ingredients. This holds true as long as each individual fraction of the polypeptide is capable of adopting an antiparallel beta-sheet structure. The tendency is that the longer chain is, the lower the alpha-beta transition temperature is, which has been linked to the presence of distorted or solvated helices with turns or beta sheets in elongating chains of poly(L-lysine). As such helical structures are apparently conducive to the alpha-beta transition, this draws a comparison to the hypothesis of metastable protein conformational states being a common stage in amyloid-formation pathways. The antiparallel architecture of the beta sheet is likely to reflect the pretransition interhelical interactions in poly(L-lysine). Namely, the chains are arranged in an antiparallel manner because of energetically favored antiparallel pre-assembly of dipolar alpha helices.     

The secondary structure of echistatin from 1H-NMR, circular-dichroism and Raman spectroscopy.

Detailed biophysical studies have been carried out on echistatin, a member of the disintegrin family of small, cysteine-rich, RGD-containing proteins, isolated from the venom of the saw-scaled viper Echis carinatus. Analysis of circular-dichroism spectra indicates that, at 20 degrees C, echistatin contains no alpha-helix but contains mostly beta-turns and beta-sheet. Two isobestic points are observed as the temperature is raised, the conformational changes associated with that observed between 40 degrees C and 72 degrees C being irreversible. Raman spectra also indicate considerable beta-turn and beta-sheet (20%) structure and an absence of alpha-helical structure. Three of the four disulphide bridges are shown to be in an all-gauche conformation, while the fourth adopts a trans-gauche-gauche conformation. The 1H-NMR spectrum of echistatin has been almost fully assigned. A single conformation was observed at 27 degrees C with the four proline residues adopting only the trans conformation. A large number of backbone amide protons were found to exchange slowly, but no segments of the backbone were found to be in either alpha-helical or beta-sheet conformation. A number of turns could be characterised. An irregular beta-hairpin contains the RGD sequence in a mobile loop at its tip. Two of the four disulphide cross-links have been identified from the NMR spectra. The data presented in this paper will serve to define the structure of echistatin more closely in subsequent studies.     

Structure and properties of phospholipid-peptide monolayers containing monomeric SP-B(1-25) II. Peptide conformation by infrared spectroscopy

The conformation and orientation of synthetic monomeric human sequence SP-B(1-25) (mSP-B(1-25)) was studied in films with phospholipids at the air-water (A/W) interface by polarization modulation infrared reflectance absorption spectroscopy (PM-IRRAS). Modified two-dimensional infrared (2D IR) correlation analysis was applied to PM-IRRAS spectra to define changes in the secondary structure and rates of reorientation of mSP-B(1-25) in the monolayer during compression. PM-IRRAS spectra and 2D IR correlation analysis showed that, in pure films, mSP-B(1-25) had a major alpha-helical conformation plus regions of beta-sheet structure. These alpha-helical regions reoriented later during film compression than beta structural regions, and became oriented normal to the A/W interface as surface pressure increased. In mixed films with 4:1 mol:mol acyl chain perdeuterated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DPPC-d(62):DOPG), the IR spectra of mSP-B(1-25) showed that a significant, concentration-dependent conformational change occurred when mSP-B(1-25) was incorporated into a DPPC-d(62):DOPG monolayer. At an mSP-B(1-25) concentration of 10 wt.%, the peptide assumed a predominantly beta-sheet conformation with no contribution from alpha-helical structures. At lower, more physiological peptide concentrations, 2D IR correlation analysis showed that the propensity of mSP-B(1-25) to form alpha-helical structures was increased. In phospholipid films containing 5 wt.% mSP-B(1-25), a substantial alpha-helical peptide structural component was observed, but regions of alpha and beta structure reoriented together rather than independently during compression. In films containing 1 wt.% mSP-B(1-25), peptide conformation was predominantly alpha-helical and the helical regions reoriented later during compression than the remaining beta structural components. The increased alpha-helical structure of mSP-B(1-25) demonstrated here by PM-IRRAS and 2D IR correlation analysis in monolayers of 4:1 DPPC:DOPG containing 1 wt.% (and, to a lesser extent, 5 wt.%) peptide may be relevant for the formation of the intermediate order 'dendritic' surface phase observed in similar surface films by epi-fluorescence.     

Environmentally induced reversible conformational switching in the yeast cell adhesion protein alpha-agglutinin

The yeast cell adhesion protein alpha-agglutinin is expressed on the surface of a free-living organism and is subjected to a variety of environmental conditions. Circular dichroism (CD) spectroscopy shows that the binding region of alpha-agglutinin has a beta-sheet-rich structure, with only approximately 2% alpha-helix under native conditions (15-40 degrees C at pH 5.5). This region is predicted to fold into three immunoglobulin-like domains, and models are consistent with the CD spectra as well as with peptide mapping and site-specific mutagenesis. However, secondary structure prediction algorithms show that segments comprising approximately 17% of the residues have high alpha-helical and low beta-sheet potential. Two model peptides of such segments had helical tendencies, and one of these peptides showed pH-dependent conformational switching. Similarly, CD spectroscopy of the binding region of alpha-agglutinin showed reversible conversion from beta-rich to mixed alpha/beta structure at elevated temperatures or when the pH was changed. The reversibility of these changes implied that there is a small energy difference between the all-beta and the alpha/beta states. Similar changes followed cleavage of peptide or disulfide bonds. Together, these observations imply that short sequences of high helical propensity are constrained to a beta-rich state by covalent and local charge interactions under native conditions, but form helices under non-native conditions.     

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).     

A 1H NMR study of human calcitonin in solution

Human calcitonin (hCT) has been investigated by NMR at 400 MHz in DMSOd6 and in an 85% DMSOd6-15% 1H2O (v/v) cryoprotective mixture. All backbone and side-chain resonances have been assigned, and the secondary structure has been determined in both solvents. In DMSOd6, the simultaneous presence of d alpha N, dNN, and some specific weak medium-range nuclear Overhauser effects, together with the amide temperature coefficients and the analysis of the NH-alpha CH spin-spin coupling constants, indicates that hCT is highly flexible but with three domains (comprising segments Asn3-Gly10, Gln14-Thr21, and Thr25-Ala31) in extended conformations which dynamically transform into isolated beta turns in the N- and C-terminal regions and into adjacent tight turns, resembling a 3(10) helix structure, in the central part. The DMSO-water mixture rigidifies the polypeptide chain, favoring an ordered, extended conformation. NOESY data indicate the presence of a short double-stranded antiparallel beta sheet in the central region made by residues 16-21 and connected by a two-residue hairpin loop formed by residues 18 and 19. Two tight turns, formed by residues 3-6 and 28-31, were also identified. The central beta sheet does not favor an amphipathic distribution of the residues as found for salmon calcitonin [Motta, A., Castiglione Morelli, M. A., Goud, N., & Temussi, P. A. (1989) Biochemistry 28, 7998-8002]. This is in agreement with the smaller tendency of hCT to form the amphipathic alpha helix, postulated to be responsible for the interaction of hCT with lipids. The possible role of the cis-trans isomerism of Pro is discussed.     

Design and synthesis of sequence-specific DNA-binding peptides

Design, synthesis and DNA binding activities of two peptides containing 32 and 102 residues are reported. A nonlinear 102-residue peptide contains four modified alpha helix-turn-alpha helix motifs of 434 cro protein. These four units are linked covalently to a carboxyterminal crosslinker containing four arms each ending with an aliphatic amino group. From CD studies we have found that in aqueous buffer in the presence of 20% trifluoroethanol the peptide residues assume alpha-helical, beta-sheet and random-coiled conformations with the alpha-helical content of about 16% at room temperature. Upon complex formation between peptide and DNA, a change in the peptide conformation takes place which is consistent with an alpha - beta transition in the DNA binding alpha helix-turn-alpha helix units of the peptide. Similar conformation changes are observed upon complex formation with the synthetic operator of a linear peptide containing residues 7-37 of 434 cro repressor. Evidently, in the complex, residues present in helices alpha 2 and alpha 3 of the two helix motif form a beta-hairpin which is inserted in the minor DNA groove. The last inference is supported by our observations that the two peptides can displace the minor groove-binding antibiotic distamycin A from poly(dA).poly(dT) and synthetic operator DNA. As revealed from DNase digestion studies, the nonlinear peptide binds more strongly to a pseudooperator Op1, located in the cro gene, than to the operator OR3. A difference in the specificity shown by the non-linear peptide and wild-type cro could be attributed to a flexibility of the linker chains between the DNA-binding domains in the peptide molecule as well as to a replacement of Thr-Ala in the peptide alpha 2-helices. Removal of two residues from the N-terminus of helix alpha 2 in each of the four DNA-binding domains of the peptide leads to a loss of binding specificity.     

Exploring the propensities of helices in PrP(C) to form beta sheet using NMR structures and sequence alignments

Neurodegenerative diseases induced by transmissible spongiform encephalopathies are associated with prions. The most spectacular event in the formation of the infectious scrapie form, referred to as PrP(Sc), is the conformational change from the predominantly alpha-helical conformation of PrP(C) to the PrP(Sc) state that is rich in beta-sheet content. Using sequence alignments and structural analysis of the available nuclear magnetic resonance structures of PrP(C), we explore the propensities of helices in PrP(C) to be in a beta-strand conformation. Comparison of a number of structural characteristics (such as solvent accessible area, distribution of (Phi, Psi) angles, mismatches in hydrogen bonds, nature of residues in local and nonlocal contacts, distribution of regular densities of amino acids, clustering of hydrophobic and hydrophilic residues in helices) between PrP(C) structures and a databank of "normal" proteins shows that the most unusual features are found in helix 2 (H2) (residues 172-194) followed by helix 1 (H1) (residues 144-153). In particular, the C-terminal residues in H2 are frustrated in their helical state. The databank of normal proteins consists of 58 helical proteins, 36 alpha+beta proteins, and 31 beta-sheet proteins. Our conclusions are also substantiated by gapless threading calculations that show that the normalized Z-scores of prion proteins are similar to those of other alpha+beta proteins with low helical content. Application of the recently introduced notion of discordance, namely, incompatibility of the predicted and observed secondary structures, also points to the frustration of H2 not only in the wild type but also in mutants of human PrP(C). This suggests that the instability of PrP(C) proteins may play a role in their being susceptible to the profound conformational change. Our analysis shows that, in addition to the previously proposed role for the segment (90-120) and possibly H1, the C-terminus of H2 and possibly N-terminus may play a role in the alpha-->beta transition. An implication of our results is that the ease of polymerization depends on the unfolding rate of the monomer. Sequence alignments show that helices in avian prion proteins (chicken, duck, crane) are better accommodated in a helical state, which might explain the absence of PrP(Sc) formation over finite time scales in these species. From this analysis, we predict that correlated mutations that reduce the frustration in the second half of helix 2 in mammalian prion proteins could inhibit the formation of PrP(Sc).     

Conformational switching and fibrillogenesis in the amyloidogenic fragment of apolipoprotein a-I

The N-terminal portion of apolipoprotein A-I corresponding to the first 93 residues has been identified as the main component of apolipoprotein A-I fibrils in a form of systemic amyloidosis. We have been able to characterize the process of conformational switching and fibrillogenesis in this fragment of apolipoprotein A-I purified directly from ex vivo amyloid material. The peptide exists in an unstructured form in aqueous solution at neutral pH. The acidification of the solution provokes a collapse into a more compact, intermediate state and the transient appearance of a helical conformation that rapidly converts to a stable, mainly beta-structure in the fibrils. The transition from helical to sheet structure occurs concomitantly with peptide self-aggregation, and fibrils are detected after 72 h. The alpha-helical conformation is induced by the addition of trifluoroethanol and phospholipids. Interaction of the amyloidogenic polypeptide with phospholipids prevents the switching from helical to beta-sheet form and inhibits fibril formation. The secondary structure propensity of the apolipoprotein A-I fragment appears poised between helix and the beta-sheet. These findings reinforce the idea of a delicate balance between natively stabilizing interactions and fatally stabilizing interactions and stress the importance of cellular localization and environment in the maintenance of protein conformation.     

Solution structure of BmKK4, the first member of subfamily alpha-KTx 17 of scorpion toxins

BmKK4 is a 30 amino acid peptide purified from the venom of the Chinese scorpion Buthus martensi Karsch. It has been classified as the first member of scorpion toxin subfamily alpha-KTx 17. The 3D structure of BmKK4 in solution has been determined by 2D NMR spectroscopy. This toxin adopts a common alpha/beta-motif, but shows a distinctive local conformation. The most novel feature is that the regular arrangements of the side chains of the residues involved in the beta-sheet of BmKK4 are distorted by a classic beta-bulge structure, which involves two residues (Asp18 and Arg19) in the first strand opposite a single residue (Tyr26) in the second strand. The bulge produces two main changes in the structure of the antiparallel beta-sheet: (1) It disrupts the normal alteration of the side chain direction; the side chain of Asp18 turns over to form a salt bridge with that of Arg19. (2) It accentuates the twist of the sheet, and alters the direction of the antiparallel beta-sheet. The unusual structural feature of the toxin is attributed to the shorter peptide segment (Leu15-Arg19) between the third and fourth Cys residues and two unique residues (Asp18 and Arg19) at the position preceding the fourth Cys. In addition, the lower affinity of the peptide for the Kv channel is correlated to the structural features: residue Arg19 instead of a Lys residue at the critical position for binding and the salt bridge formed between residues Arg19 and Asp18.     

Glucagon antagonists: contribution to binding and activity of the amino-terminal sequence 1-5, position 12, and the putative alpha-helical segment 19-27

Glucagon binding to and recognition by its cell surface receptor is the necessary first step in the cascade of events leading to the activation of adenylate cyclase by the hormone. It has long been presumed that glucagon adopts an ordered conformation upon binding to its membrane-bound receptor. A recent model of this three-dimensional structure based on biophysical data, predicts beta-turns at positions 2-5, 10-13, and 15-18, and an alpha-helical region between residues 19-27. Our approach in the design of antagonists of glucagon was to elucidate the steric and electronic features that stabilize these secondary structures to obtain analogs that bind with high affinity to the receptor but do not activate adenylate cyclase. Nineteen glucagon analogs incorporating structural changes at the amino-terminal sequence 1-5, at positions 9 and 12, and at the carboxyl-terminal helical region were synthesized. Des-His1-[Glu9]glucagon amide was recently shown to be a competitive inhibitor. Our synthetic studies in combination with this modification have resulted in seven new glucagon antagonists. The implications for the structural and conformational properties required for binding and activity of glucagon and the glucagon peptide family are discussed.     

Recurrent alpha beta loop structures in TIM barrel motifs show a distinct pattern of conserved structural features.

A systematic survey of seven parallel alpha/beta barrel protein domains, based on exhaustive structural comparisons, reveals that a sizable proportion of the alpha beta loops in these proteins--20 out of a total of 49--belong to either one of two loop types previously described by Thornton and co-workers. Six loops are of the alpha beta 1 type, with one residue between the alpha-helix and beta-strand, and 13 are of the alpha beta 3 type, with three residues between the helix and the strand. Protein fragments embedding the identified loops, and termed alpha beta connections since they contain parts of the flanking helix and strand, have been analyzed in detail revealing that each type of connection has a distinct set of conserved structural features. The orientation of the beta-strand relative to the helix and loop portions is different owing to a very localized difference in backbone conformation. In alpha beta 1 connections, the chain enters the beta-strand via a residue adopting an extended conformation, while in alpha beta 3 it does so via a residue in a near alpha-helical conformation. Other conserved structural features include distinct patterns of side chain orientation relative to the beta-sheet surface and of main chain H-bonds in the loop and the beta-strand moieties. Significant differences also occur in packing interactions of conserved hydrophobic residues situated in the last turn of the helix. Yet the alpha-helix surface of both types of connections adopts similar orientations relative to the barrel sheet surface. Our results suggest furthermore that conserved hydrophobic residues along the sequence of the connections, may be correlated more with specific patterns of interactions made with neighboring helices and sheet strands than with helix/strand packing within the connection itself. A number of intriguing observations are also made on the distribution of the identified alpha beta 1 and alpha beta 3 loops within the alpha/beta-barrel motifs. They often occur adjacent to each other; alpha beta 3 loops invariably involve even numbered beta-strands, while alpha beta 1 loops involve preferentially odd beta-strands; all the analyzed proteins contain at least one alpha beta 3 loop in the first half of the eightfold alpha/beta barrel. Possible origins of all these observations, and their relevance to the stability and folding of parallel alpha/beta barrel motifs are discussed.     

Dissecting the assembly of Abeta16-22 amyloid peptides into antiparallel beta sheets

Multiple long molecular dynamics simulations are used to probe the oligomerization mechanism of Abeta(16-22) (KLVFFAE) peptides. The peptides, in the monomeric form, adopt either compact random-coil or extended beta strand-like structures. The assembly of the low-energy oligomers, in which the peptides form antiparallel beta sheets, occurs by multiple pathways with the formation of an obligatory alpha-helical intermediate. This observation and the experimental results on fibrillogenesis of Abeta(1-40) and Abeta(1-42) peptides suggest that the assembly mechanism (random coil --> alpha helix --> beta strand) is universal for this class of peptides. In Abeta(16-22) oligomers both interpeptide hydrophobic and electrostatic interactions are critical in the formation of the antiparallel beta sheet structure. Mutations of either hydrophobic or charged residues destabilize the oligomer, which implies that the 16-22 fragments of Arctic (E22G), Dutch (E22Q), and Italian (E22K) mutants are unlikely to form ordered fibrils.     

Conformation of the third domain of turkey ovomucoid in solution. Structural analysis by two-dimensional Overhauser nuclear effect spectroscopy]

The conformations of a polypeptide chain of turkey ovomucoid third domain and its modified form with split reactive site peptide bond Leu-18--Glu-19 have been determined by the literary two-dimensional nuclear Overhauser effect spectroscopy data using an earlier suggested method. It has been found that the polypeptide domain backbone contains an alpha-helical fragment (residues 32-47), five segments having extended conformation (1-5, 11-17, 19-25, 29-31, 48-50) and beta-turn type 1 (26-29). Segments 23-26, 28-31 and 50-51 form an antiparallel beta-structure. Conformational states of the residues entering irregular domain segments have been analysed. Splitting of the reactive site peptide bond Leu-18--Glu-19 is shown to cause insignificant changes in the conformations of a number of amino acid residues except for Val-6 and Asp-7 ones which undergo essential conformational alterations. The conformations of domain in solution and of japanese quail ovomucoid third domain in crystal have been compared. The root-mean-square deviations for phi and psi angles indicate their high similarity. The conformations of turkey ovomucoid third domain and proteinase inhibitor BUSI IIA in solution have been analysed. In spite of moderate (50%) homology of primary structures, some 75% of amino acid residues are shown to have close conformational phi and psi parameters.     

Structural analysis of amyloid beta peptide fragment (25-35) in different microenvironments

Amyloid beta (Abeta) peptides are one of the classes of amphiphilic molecules that on dissolution in aqueous solvents undergo interesting conformational transitions. These conformational changes are known to be associated with their neuronal toxicity. The mechanism of structural transition involved in the monomeric Abeta to toxic assemblage is yet to be understood at the molecular level. Early results indicate that oriented molecular crowding has a profound effect on their assemblage formation. In this work, we have studied how different microenvironments affect the conformational transitions of one of the active amyloid beta-peptide fragments (Abeta(25-35)). Spectroscopic techniques such as CD and Fourier transform infrared spectroscopy were used. It was observed that a stored peptide concentrates on dissolution in methanol adopts a minor alpha-helical conformation along with unordered structures. On changing the methanol concentration in the solvated film form, the conformation switches to the antiparallel beta-sheet structure on the hydrophilic surface, whereas the peptide shows transition from a mixture of helix and unordered structure into predominantly a beta-sheet with minor contribution of helix structure on the hydrophobic surface. Our present investigations indicate that the conformations induced by the different surfaces dictate the gross conformational preference of the peptide concentrate.     

Alanine-scanning mutagenesis of the beta-sheet region of phage T4 lysozyme suggests that tertiary context has a dominant effect on beta-sheet formation

In general, alpha-helical conformations in proteins depend in large part on the amino acid residues within the helix and their proximal interactions. For example, an alanine residue has a high propensity to adopt an alpha-helical conformation, whereas that of a glycine residue is low. The sequence preferences for beta-sheet formation are less obvious. To identify the factors that influence beta-sheet conformation, a series of scanning polyalanine mutations were made within the strands and associated turns of the beta-sheet region in T4 lysozyme. For each construct the stability of the folded protein was reduced substantially, consistent with removal of native packing interactions. However, the crystal structures showed that each of the mutants retained the beta-sheet conformation. These results suggest that the structure of the beta-sheet region of T4 lysozyme is maintained to a substantial extent by tertiary interactions with the surrounding parts of the protein. Such tertiary interactions may be important in determining the structures of beta-sheets in general.     

The secondary structure of peptides derived from caseins: a circular dichroism study

Three peptides have been formed by proteolytic digestion of individual casein proteins and their secondary structures characterised by far-UV circular dichroism (CD). Peptide alpha s1(1-23), residues 1-23 of alpha s1-casein, was generated by treatment of the parent protein with chymosin. Peptides beta(1-28) and beta(1-52), residues 1-28 and 1-52 of beta-casein, were plasmin- and chymotrypsin-generated fragments, respectively. Analysis of the CD spectra revealed that in aqueous solution all three peptides have secondary structures composed exclusively of beta-sheet and random coil. A limited amount of alpha-helix was formed in two of the three peptides upon treatment with high concentrations (greater than 40% (v/v] of 2,2,2-trifluoroethanol. Partial dephosphorylation (60%) of beta(1-28) and beta(1-52) by treatment with alkaline phosphatase resulted in homogeneous preparations, as judged by polyacrylamide gel electrophoresis, which exhibited increased hydrophobicity. This reduction in the level of phosphorylation of serine residues 15, 17, 18 and 19 led to increased propensity for helix formation in the peptides in the presence of 2,2,2-trifluoroethanol, but no alpha-helical structures were detected in the dephosphorylated peptides in the absence of 2,2,2-trifluoroethanol.