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).     

The effect of Abeta conformation on the metal affinity and aggregation mechanism studied by circular dichroism spectroscopy

The conformational change and associated aggregation of beta amyloid (Abeta) with or without metals is the main cause of Alzheimer's disease (AD). In order to further understand the effects of Abeta and its associated metals on the aggregation mechanism, the influence of Abeta conformation on the metal affinity and aggregation was investigated using circular dichroism (CD) spectroscopy. The Abeta conformation is dependent on pH and trifluoroethanol (TFE). The binding of metals to Abeta was found to be dependent on the Abeta conformation. The aggregation induced by Abeta itself or its associated metals is completely diminished for Abeta in 40% TFE. Only in 5% and 25% TFE can Abeta undergo an alpha-helix to beta-sheet aggregation, which involve a three-state mechanism for the metal-free state, and a two-state transition for the metal-bound state, respectively. The aggregation-inducing activity of metals is in the order, Cu2+ > Fe3+ > or = Al3+ > Zn2+.     

Fluoroalcohols induced unfolding of Succinylated Con A: native like beta-structure in partially folded intermediate and alpha-helix in molten globule like state

Concanavalin A (Con A) exists in dimeric state at pH 5. In concentration range 20-60% (v/v) 2,2,2-trifluoroethanol (TFE) and 2-40% (v/v) 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), Con A at pH 5.0 shows visible aggregation. However, when succinyl Con A was used, no aggregation was observed in the entire concentration range of fluoroalcohols (0-90% v/v TFE and HFIP) and resulted in stable alpha-helix formation. Temperature-induced concentration-dependent aggregation in Con A was also found to be prevented/reduced in succinylated form. Possible role of electrostatic repulsion among residues in the prevention of hydrophobically driven aggregation has been discussed. Results indicate that succinylation of a protein resulted in greater stability (in both beta-sheet and alpha-helical forms) against alcohol-induced and temperature-induced concentration-dependent aggregation and this observation may play significant role in amyloid-forming proteins. Effect of TFE and HFIP on the conformation of a dimeric protein, Succinylated Con A, has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of hydrophobic dye ANS (8-anilinonaphthalene-1-sulfonic acid). Far UV-CD, a probe for secondary structure shows loss of native secondary structure in the presence of low concentration of both the alcohols, TFE (10% v/v) and HFIP (4% v/v). Upon addition of higher concentration of these alcohols, Succinylated Con A exhibited transformation from beta-sheet to alpha-helical structure. Intrinsic tryptophan fluorescence studies, ANS binding and near UV-CD experiments indicate the protein is more expanded, have more exposed hydrophobic surfaces and highly disrupted tertiary structure at 60% (v/v) TFE and 30% (v/v) HFIP concentrations. Taken together, these results it might be concluded that TFE and HFIP induce two intermediate states at their low and high concentrations in Succinyl Con A.     

Conformational changes and inactivation of bovine carbonic anhydrase II in 2,2,2-trifluoroethanol solutions

Changes in unfolding and enzymatic activity of bovine carbonic anhydrase II (BCA II) in different concentrations of 2,2,2-trifluoroethanol (TFE) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) fluorescence emission spectra, far-UV CD spectra, and enzyme activity. The results showed that the activity and conformation of BCA II changed according to the concentration of TFE. Significant aggregation was observed when BCA II was denatured at TFE concentrations between 10 and 35% (v/v). When the concentration of TFE exceeded 40%, the aggregation of BCA II was not very obvious. The activity of BCA II decreased almost to zero as the TFE concentration reached 26%. The ANS fluorescence spectra indicated the tertiary conformations of BCA II were more stable in solutions with TFE concentrations lower than 15% (v/v) and higher than 40% (v/v). Far-UV CD spectra showed that high concentrations (higher than 25%) of TFE could induce BCA II to form more alpha-helix structures and caused these structures to be in relatively stable states. The native conformation of BCA II being destroyed after its inactivity indicated that the active sites of BCA II is situated in a limited region and has more flexibility than the whole enzyme molecule.     

Effect of organic solvents on the molten globule state of procerain: beta-sheet to alpha-helix switchover in presence of trifluoroethanol

The effect of methanol and trifluoroethanol (TFE) on the structure and folding of molten globule state of procerain, a cysteine protease from Calotropis procera, was studied by circular dichroism spectroscopy. The magnitude of ellipticity at 215 nm, as a measure of beta-sheet content, is dependent on the concentration of the TFE. Interestingly, a switch over from the beta-sheet structure of the molten globule state to alpha-helix was observed at 60% TFE and the ellipticity at 222 nm increased as a function of TFE concentration beyond this critical TFE concentration. Temperature induced unfolding of the molten globule state of procerain in 10% methanol showed stabilization of alpha-rich domain with concomitant destabilization of beta-rich domain. Using higher concentration of methanol (20-40 %) had no stabilizing effect on the alpha-rich domain however, the beta-rich domain was destabilized, indicating that the stability of the domains were not interdependent and that a low concentration of methanol induced stabilization in alpha-rich domain.     

Low-ultraviolet circular dichroism spectroscopy of sequential peptides 1-63, 64-95, 96-128, and 129-168 derived from myelin basic protein of rabbit

Four sequential peptides (sequences 1-63, 64-95, 96-128, and 129-168) derived from rabbit myelin basic protein by thrombic cleavage were examined by low-ultraviolet circular dichroism spectroscopy in 0.5 mM tris(hydroxymethyl)aminomethane hydrochloride (pH approximately 7.2) containing 0-92% trifluoroethanol (TFE). In the absence of the alcohol, all of the peptides contained a significant amount (17-29%) of beta-structure. In the presence of relatively low concentrations (up to 30%) of TFE, all of the peptides except 96-128 adopted considerable alpha-helix (16-33%). This involved a transition from the beta-structure in peptide 1-63 and transitions from the nonordered structure in peptides 1-63, 64-95, and 129-168. Furthermore, additional alpha-helix formed in peptide 1-63 between 30% and 92% TFE at the expense of nonordered structure, whereas the alpha-helix formation above 50% TFE in peptide 129-168 resulted largely from a beta-structure----alpha-helix transition. With the exception of the 129-168 peptide, approximately 65-100% of the maximum level of beta-structure persisted throughout the entire range of TFE concentration. In the case of peptide 129-168, however, most of the beta-structure was converted to alpha-helix and nonordered structure at 75% TFE. While the present results support our previous assignments of beta-structure- and alpha-helix-forming regions to specific amino acid sequences of the basic protein, they also demonstrate that the beta-structure----alpha-helix transitions evidenced at various concentrations of TFE were influenced to a considerable degree by the length of the peptide, presumably due to the presence or absence of interactions between noncontiguous portions of the myelin basic protein polypeptide chain.     

Amyloid fibril formation by peptide LYS (11-36) in aqueous trifluoroethanol

Peptide LYS (11-36), derived from the beta-sheet region of T4 lysozyme, forms an amyloid fibril in aqueous trifluoroethanol (TFE) at elevated temperature. The peptide has a moderate alpha-helix content in 20 and 50% (v/v) TFE solution; large quantities of fibrils were formed after incubation at 55 degrees C for 2 weeks as monitored by a thioflavin T fluorescence assay. No fibrils were observed when the peptide initially existed predominantly as a random coil or as a complete alpha helix. Our results suggest that a moderate amount of alpha helix and random coil present in the peptide initially facilitates the fibril-formation process, but a high alpha-helix content inhibits fibril formation. Transmission electron microscopy revealed several types of fibril morphologies at different TFE concentrations. The fibrils were highly twisted and consisted of interleaved protofilaments in 50% TFE, while smooth and flat ribbonlike fibrils were found in 20% TFE. In 50% TFE, the fibril growth rate of LYS (11-36) was found to depend strongly on peptide concentration and seeding but was insensitive to solution pH and ionic strength.     

Biphasic effects of alcohols on the phase transition of poly(L-lysine) between alpha-helix and beta-sheet conformations.

Poly(L-lysine) exists as a random-coil at neutral pH, an alpha-helix at alkaline pH, and a beta-sheet when the alpha-helix poly(L-lysine) is heated. The present Fourier-transform infrared (FTIR) study showed that short-chain alcohols (methanol, ethanol, and 2-propanol) partially transformed alpha-helix poly(L-lysine) to beta-sheet when their concentrations were low. At higher concentrations, however, these alcohols reversed the reaction, and the alcohol-induced beta-sheet was transformed back to alpha-helix structure. The reversal occurred at 1.40 M methanol, 0.96 M ethanol, and 0.55 M 2-propanol. The alcohol effects on the secondary structure were further investigated by circular dichroism (CD) on the thermally induced beta-sheet poly(L-lysine). Methanol, ethanol, and 1-propanol, but not 1-butanol, shifted the negative mean-residue ellipticity at 217 nm of the beta-sheet poly(L-lysine) to the positive side at low concentrations of the alcohols and to the negative side at high concentrations. With 1-butanol, only the positive-side shift was observed. The positive-side shift at low concentrations of alcohols indicates enhancement of the hydrophobic interactions among the side chains of the polypeptide in the beta-sheet conformation. The negative-side shift indicates a partial transformation to alpha-helix. The shift from the positive to negative side occurred at 7.1 M methanol, 4.6 M ethanol, and 3.1 M 1-propanol. The alcohol concentrations for the beta-to-alpha transition were higher in the CD study than in the IR study.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational studies of alanine-rich peptide using CD and FTIR spectroscopy.

The circular dichroism (CD) and Fourier transform infrared (FTIR) methods were applied to the conformational studies of alanine-rich peptide Ac-K-[A]11-KGGY-NH2 (where K is lysine, A is alanine, G is glycine and Y is tyrozyne) in water, methanol (MeOH) and trifluoroethanol (TFE). The analysis of CD-spectra of the peptide in water at different concentrations revealed that the secondary structure content depends on the peptide concentration and pH of the solution. The increase of the peptide concentration causes a decrease of alpha-helix content and, simultaneously, an increase of beta-sheet structure, while the unordered structure is the predominant one. Additional elements are discovered in MeOH and TFE but alpha-helix and beta-turns predominate. Moreover, in these solutions the percentage content of the secondary structure does not depend on the temperature. FTIR measurements, carried out at higher peptide concentration (about one order of magnitude) than these CD measurements mentioned above, revealed that in water solution the solid state beta-sheet, and aggregated structures, dominate. However, in TFE the most abundant are alpha-helix and beta-turns structures. The thioflavine T assay showed the tendency of the studied peptide for aggregate.     

Effect of trifluoroethanol on the conformational stability of a hyperthermophilic esterase: a CD study

The conformational stability of the hyperthermophilic esterase AFEST from Archeoglobus fulgidus against the denaturing action of 2,2,2-trifluoroethanol (TFE) has been investigated by means of circular dichroism (CD) measurements. At room temperature far-UV and near-UV CD spectra point out the occurrence of a co-operative transition from the native structure to a denatured state characterized by a high content of alpha-helix. The TFE concentration at half-completion of the transition proves to be 3.5 M (25% v v(-1)), by recording the molar ellipticity at both 222 and 276 nm. Thermal transition curves of AFEST in the absence and in the presence of TFE indicate a significant stability decrease on increasing the TFE concentration. The denaturation temperature is 99 degrees C for native AFEST, but becomes 85 degrees C at 1.4 M TFE (10% v v(-1)), and 56 degrees C at 2.8 M TFE (20% v v(-1)). It is also shown that, even though AFEST is very resistant to temperature, its resistance towards the denaturing action of TFE is similar to that of mesophilic proteins, including an esterase from Escherichia coli, AES. The proposal of a general mechanism for the TFE action on globular proteins leads to a reliable rationale of experimental data.     

The alpha-helix to beta-sheet transition in poly(L-lysine): effects of anesthetics and high pressure.

Poly(L-lysine) exists in a random-coil formation at a low pH, alpha-helix at a pH above 10.6, and transforms into beta-sheet when the alpha-helix polylysine is heated. Each conformation is clearly distinguishable in the amide-I band of the infrared spectrum. The thermotropic alpha-to-beta transition was studied by using differential scanning calorimetry. At pH 10.6, the transition temperature was 43.5 degrees C and the transition enthalpy was 170 cal/mol residue. At pH 11.85, the measurements were 36.7 degrees C and 910 cal/mol residue, respectively. Volatile anesthetics (chloroform, halothane, isoflurane and enflurane) partially transformed alpha-helix polylysine into beta-sheet. The transformation was reversed by the application of hydrostatic pressure in the range of 100-350 atm. Apparently, the alpha-to-beta transition was induced by anesthetics through partial dehydration of the peptide side-chains (beta-sheet surface is less hydrated than alpha-helix). High pressure reversed this process by re-hydrating the peptide. Because the membrane spanning domains of channel and receptor proteins are predominantly in the alpha-helix conformation, anesthetics may suppress the activity of excitable cells by transforming them into a less than optimal structure for electrogenic ion transport and neurotransmission. Proteins and lipid membranes maintain their structural integrity by interaction with water. That which attenuates the interaction will destabilize the structure. These data suggest that anesthetics alter macromolecular conformations essentially by a solvent effect, thereby destroying the solvation water shell surrounding macromolecules.     

Mechanism by which the amyloid-like fibrils of a beta 2-microglobulin fragment are induced by fluorine-substituted alcohols

Although the formation of an alpha-helix or partial unfolding of proteins has been suggested to be important for amyloid fibrils to form in alcohols, the exact mechanism involved remains elusive. To obtain further insight into the development of amyloid fibrils, we used a 22-residue peptide, K3, corresponding to Ser20 to Lys41 of intact beta2-microglobulin. Although K3 formed an alpha-helix at high concentrations of 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) in 10 mM HCl (pH approximately 2), the helical content was not high, indicating a low preference to do so. The partly alpha-helical conformation was converted with time into a highly ordered beta-sheet with a fibrillar morphology as revealed by atomic force microscopy. Importantly, the TFE and HFIP-induced fibrillation exhibited a concentration dependence with a maximum at approximately 20 and approximately 10% (v/v), respectively, slightly below the concentrations at which these alcohols form dynamic clusters. Focusing on the similarity of the effects of alcohol on proteins with those of sodium dodecyl sulfate (SDS), we examined the effects of SDS on K3. SDS also induced fibrils to form with a maximum at approximately 4 mM, slightly below the critical micelle concentration. These results indicate that, with an increase in the concentration of hydrophobic cosolvent (TFE, HFIP, or SDS), a delicate balance of decreasing hydrophobic interactions and increasing polar interactions (i.e. H-bonds) in and between peptides leads to the formation of ordered fibrils with a bell-shaped concentration dependence.     

Characterization of a common intermediate of pea lectin in the folding pathway induced by TFE and HFIP

When pea lectin was exposed to a low pH range, it was found that the secondary structure of the lectin resisted conformational changes to a large extent up to pH 2.4 and below this pH, a sharp transition was observed which could be due to the presence of 27 acidic amino acid residues present in the protein. The effects of 1,1,1,3,3,3 hexafluoro-isopropanol (HFIP) and 2,2,2-Trifluoroethanol (TFE) on the conformation of pea lectin at pH 2.4 were studied using circular dichroism and fluorescence spectroscopy. Analysis varying the TFE concentration showed that up to 80% TFE (v/v) protein retained the residual beta-structure accompanied by a loss in tertiary structure. A similar conformation is presumed to exist at 4% HFIP (v/v), with an increase in HFIP concentration structural rearrangements occurred and a transition from beta-structure to alpha-helical structure started from 12% HFIP which completed at 30% HFIP. Our studies show the occurrence of a common intermediate in the folding pathway of pea lectin induced by two different fluoroalcohols, which differ in their mode of action to stabilize the secondary structure of a given protein. While TFE was not found to induce any alpha-helical structure, HFIP caused the transition of pea lectin, which is predominantly a beta-sheet protein, to a structure rich in alpha-helical contacts. Thus, our results also point out the possibility of a non-hierarchical model of protein folding in lectins.     

Conformational changes and inactivation of bovine carbonic anhydrase II in 2,2,2-Trifluoroethanol solutions

Changes in unfolding and enzymatic activity of bovine carbonic anhydrase II (BCA II) in different concentrations of 2,2,2-trifluoroethanol (TFE) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) fluorescence emission spectra, far-UV CD spectra, and enzyme activity. The results showed that the activity and conformation of BCA II changed according to the concentration of TFE. Significant aggregation was observed when BCA II was denatured at TFE concentrations between 10 and 35% (v/v). When the concentration of TFE exceeded 40%, the aggregation of BCA II was not very obvious. The activity of BCA II decreased almost to zero as the TFE concentration reached 26%. The ANS fluorescence spectra indicated the tertiary conformations of BCA II were more stable in solutions with TFE concentrations lower than 15% (v/v) and higher than 40% (v/v). Far-UV CD spectra showed that high concentrations (higher than 25%) of TFE could induce BCA II to form more α-helix structures and caused these structures to be in relatively stable states. The native conformation of BCA II being destroyed after its inactivation indicated that the active site of BCA II is situated in a limited region and has more flexibility than the whole enzyme molecule.     

Formation of an alpha-helix in human tumor necrosis factor-alpha by guanidine hydrochloride-induced unfolding

Human tumor necrosis factor-alpha (TNF-alpha) is a trimeric protein consisting primarily of beta-sheet. GdnHCl-induced unfolding of TNF-alpha was investigated at room temperature by circular dichroism (CD) and size exclusion chromatography. The secondary and tertiary structure of TNF-alpha persisted up to 0.9N GdnHCl regardless of incubation time, but, in the range of 1.2 N to 2.1 N GdnHCl, there was loss of tertiary structure accompanied by the formation of an alpha-helix, as revealed by far- and near-UV CD spectra. The structural changes occurred gradually in 1.2 and 2.1 N GdnHCl, but were rapid in 1.5 and 1.8 N GdnHCl. The GdnHCl-induced state of TNF-alpha is an unfolded, alpha-helical aggregate of about 130 monomers, as shown by size exclusion chromatography. We suggest the most likely pathway for the transition from beta-sheet to alpha-helix.     

Low-ultraviolet circular dichroism spectroscopy of oligopeptides 1-95 and 96-168 derived from myelin basic protein of rabbit

Myelin basic protein (MBP) is a major protein constituent of the myelin sheath of the central nervous system, where it is believed to have functional alpha-helical segments. One element of the function of the protein might be "conformational adaptability" of specific regions of its amino acid sequence, since the purified protein appears to be largely devoid of ordered structure. To pursue this question, low-ultraviolet circular dichroism (CD) spectroscopy was conducted on the sequential thrombic peptides 1-95 and 96-168 of the protein in the presence of 0-92% trifluoroethanol (TFE), a solvent known to promote stable secondary structures in polypeptides. The series of CD spectra of the oligopeptides were subjected to a computerized best-fit analysis of four peptide conformations, the alpha-helix, beta-structure, beta-turn, and nonordered form. Agreement between experimental and best-fit composite spectra was achieved when standard CD curves of peptide conformations were derived from known theoretical spectra and experimental spectra of polypeptides. In dilute buffer alone, oligopeptides 1-95 and 96-168 evidence no alpha-helix but significant beta-structure (18% and 23%, respectively), as well as a predominant, extended nonordered conformation. However, the two parts of the protein differed in conformational adaptability. From 0% to 30% TFE, 96-168 exhibited concomitant transitions to 10% helix and 32% beta-structure from the nonordered form. In contrast, in 10-30% TFE, 1-95 underwent a transition to approximately 21% helix with partial loss of beta-structure as well as nonordered form; higher concentrations of TFE (40-75%) promoted additional transitions to both helix and beta-structure (totaling 33% and 25%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

The complex structure transition of Humanin peptides by sodium dodecylsulfate and trifluoroethanol

We have examined the structure of two Humanin (HN) analog peptides, HNG and AGA-(C8R)HNG17, in the presence of sodium dodecylsulfate (SDS) and trifluoroethanol (TFE) using CD and sedimentation velocity. Both HNG and AGA-(C8R)HNG17 underwent complex conformational changes with increasing concentrations of SDS and TFE, in contrast to general trend of increasing alpha-helix with their concentration. To our surprise, both peptides appear to converge into a similar structure in SDS and TFE at higher concentrations; e.g., above 0.05 % SDS or 30-40 % TFE. Sedimentation velocity analysis showed extensive aggregation of HNG at 0.1 mg/ml in PBS in the absence of SDS, but a highly homogeneous solution in 0.1 % SDS, indicating formation of a uniform structure by SDS. These two peptides also formed an intermediate structure both in SDS and TFE at lower concentrations, which appeared to be associated with extensive aggregation. It is interesting that the structure changes of these peptides occur well below the critical micelle concentration of SDS, suggesting that conformational changes are mediated through molecular, not micellar, interactions with SDS.     

Evidence for an alpha-helix --> pi-bulge helicity modulation for the neu/erbB-2 membrane-spanning segment. A 1H NMR and circular dichroism study

The 35-residue peptide corresponding to the very hydrophobic transmembrane region of the tyrosine kinase receptor neu, Neu(TM35), has been synthesized. The peptide can be solubilized in millimolar concentrations in TFE or incorporated into an SDS-water micellar solution or into well-hydrated DMPC/DCPC bicelles. In all these media, circular dichroism demonstrated that the peptide adopts a helical structure for about 80% of its amino acids. The peptide is monomeric below 2 mM in TFE, as also determined by variable concentration experiments. The three-dimensional solution structure in TFE has been obtained by homonuclear proton NMR and shows a well-defined alpha-helix from residues 4 to 21, then a pi-bulge from Ile(22) to Gly(28), and a final short alpha-helix from positions 29 to 32. This experimental finding is in agreement with structures predicted recently by molecular dynamics calculations in a vacuum [Sajot, N., and Genest, M. (2000) Eur. Biophys. J. 28, 648-662]. The biological implications of a possible retention of this structure in a membrane environment are finally discussed.     

Secondary structure of Escherichia coli glucosamine-6-phosphate deaminase from amino acid sequence and circular dichroism spectroscopy

The secondary structure of the purified glucosamine-6-phosphate deaminase from Escherichia coli K12 was investigated by both circular dichroism (CD) spectroscopy and empirical prediction methods. The enzyme was obtained by allosteric-site affinity chromatography from an overproducing strain bearing a pUC18 plasmid carrying the structural gene for the enzyme. From CD analysis, 34% of alpha-helix, 9% of parallel beta-sheet, 11% of antiparallel beta-sheet, 15% turns and 35% of non-repetitive structures, were estimated. A joint prediction scheme, combining six prediction methods with defined rules using several physicochemical indices, gave the following values: alpha-helix, 37%; beta-sheet, 22%; turns, 18% and coil, 23%. The structure predicted showed also a considerable degree of alternacy of alpha and beta structures; 64% of helices are amphipathic and 90% of beta-sheets are hydrophobic. Overall, the data suggest that deaminase has as dominant motif, an alpha/beta structure.     

Protein structural perturbation and aggregation on homogeneous surfaces

We have demonstrated that globular proteins, such as hen egg lysozyme in phosphate buffered saline at room temperature, lose native structural stability and activity when adsorbed onto well-defined homogeneous solid surfaces. This structural loss is evident by alpha-helix to turns/random during the first 30 min and followed by a slow alpha-helix to beta-sheet transition. Increase in intramolecular and intermolecular beta-sheet content suggests conformational rearrangement and aggregation between different protein molecules, respectively. Amide I band attenuated total reflection/Fourier transformed infrared (ATR/FTIR) spectroscopy was used to quantify the secondary structure content of lysozyme adsorbed on six different self-assembled alkanethiol monolayer surfaces with -CH3, -OPh, -CF3, -CN, -OCH3, and -OH exposed functional end groups. Activity measurements of adsorbed lysozyme were in good agreement with the structural perturbations. Both surface chemistry (type of functional groups, wettability) and adsorbate concentration (i.e., lateral interactions) are responsible for the observed structural changes during adsorption. A kinetic model is proposed to describe secondary structural changes that occur in two dynamic phases. The results presented in this article demonstrate the utility of the ATR/FTIR spectroscopic technique for in situ characterization of protein secondary structures during adsorption on flat surfaces.