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.     

Intermediacy of poly(L-proline) II and beta-strand conformations in poly(L-lysine) beta-sheet formation probed by temperature-jump/UV resonance Raman spectroscopy

Ultraviolet resonance Raman spectroscopy (UVRR) in combination with a nanosecond temperature jump (T-jump) was used to investigate early steps in the temperature-induced alpha-helix to beta-sheet conformational transition of poly(L-lysine) [poly(K)]. Excitation at 197 nm from a tunable frequency-quadrupled Ti:sapphire laser provided high-quality UVRR spectra, containing multiple conformation-sensitive amide bands. Although un-ionized poly(K) (pH 11.6) is mainly alpha-helical below 30 degrees C, there is a detectable fraction (approximately 15%) of unfolded polypeptide, which is mainly in the poly(L-proline) II (PPII) conformation. However, deviations from the expected amide I and II signals indicate an additional conformation, suggested to be beta-strand. Above 30 degrees C un-ionized poly(K) forms a beta-sheet at a rate (minutes) which increases with increasing temperature. A 22-44 degrees C T-jump is accompanied by prompt amide I and II difference signals suggested to arise from a rapid shift in the PPII/beta-strand equilibrium. These signals are superimposed on a subsequently evolving difference spectrum which is characteristic of PPII, although the extent of conversion is low, approximately 2% at the 3 micros time limit of the experiment. The rise time of the PPII signals is approximately 250 ns, consistent with melting of short alpha-helical segments. A model is proposed in which the melted PPII segments interconvert with beta-strand conformation, whose association through interstrand H-bonding nucleates the formation of beta-sheet. The intrinsic propensity for beta-strand formation could be a determinant of beta-sheet induction time, with implications for the onset of amyloid diseases.     

Temperature-induced conformational changes in amyloid beta(1-40) peptide investigated by simultaneous FT-IR microspectroscopy with thermal system

Temperature-dependent secondary structures of the amyloid beta(1-40) peptide in the solid state were studied by simultaneous Fourier transform infrared/differential scanning calorimetry (FT-IR/DSC) microspectroscopic system with the heating-cooling-reheating cycle. The result indicates that a thermal transition temperature at 45 degrees C was easily obtained from the three-dimensional plot of the transmission FT-IR spectra as a function of temperature. Furthermore, the thermal-dependent conformational transformations, due to denaturation and aggregation, of solid amyloid beta(1-40) were mainly evidenced by reducing the compositions from 37 to 20-24% for alpha-helical and random coil structures but increasing the components from 27 to 45% for intermolecular beta-sheet structures. Thermal-irreversible behavior and a poor thermal stability of solid amyloid beta(1-40) were also observed from the poor restoration of the secondary conformational changes in the heated sample.     

Secondary structure changes stabilize the reactive-centre cleaved form of SERPINs. A study by 1H nuclear magnetic resonance and Fourier transform infrared spectroscopy.

Proteinase inhibitor members of the SERPIN superfamily are characterized by the presence of a proteolytically sensitive reactive-site loop. Cleavage within this region results in a conformational transition from an unstable "stressed" native protein to a more stable "relaxed" cleaved molecule. In order to identify the principal molecular aspects of this transition, 1H nuclear magnetic resonance (n.m.r.) and FT-IR spectroscopy were applied to the study of four SERPINs. 1H n.m.r. spectra of approximately 20 high-field ring-current-shifted methyl signals exhibited slightly different chemical shifts in the native and cleaved forms of alpha 1-antitrypsin (alpha 1-AT), alpha 1-antichymotrypsin (alpha 1-ACT) and C1 inhibitor (C1-INH), but not ovalbumin, between 20 degrees C and 90 degrees C. Ring current calculations based on crystal co-ordinates for cleaved alpha 1-AT and alpha 1-ACT and native ovalbumin showed that these signals originate from highly localized interactions between different buried residues corresponding to alpha-helix and beta-sheet segments of the SERPIN fold. The small shift changes correspond to small relative conformational side-chain rearrangements of about 0.01 nm to 0.05 nm in the protein hydrophobic core, i.e. the tertiary structure interactions in the two forms of the SERPIN fold are well-preserved, and changes in this appear unimportant for the stabilization found after reactive centre cleavage. Fourier transform infrared (FT-IR) spectroscopic studies of the amide I band showed that the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH contain 28% to 36% alpha-helix and 38% to 44% beta-sheet. Second derivative FT-IR spectra using H2O and 2H2O buffers revealed very large differences in the amide I band between the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH, but not for ovalbumin. The alpha-helix band was most sensitive to 1H-2H exchange, while the beta-sheet bands were not, and greater amounts of antiparallel beta-sheet were detected in the cleaved form. 1H n.m.r. showed that polypeptide amide 1H-2H exchange was greater in the native forms of alpha 1-AT, alpha 1-ACT and C1-INH than in their cleaved forms, whereas for ovalbumin it was unchanged. The FT-IR and 1H-2H exchange data show that alterations in the secondary structure are central to the stabilization of the cleaved SERPIN structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thermal-induced changes in the secondary conformation of superoxide dismutase containing different metal ions

The thermal stability of three superoxide dismutases (SODs) with different metal ions (Mn, Cu/Zn, Fe) in the solid state was studied by a Fourier transform infrared (FT-IR) microspectroscopy combined with thermal analyzer. The IR spectra showed a maximum peak at 1652 cm(-1) for all the native SODs in the amide I band, suggesting a predominant random coil with less alpha-helix structures. By heating each sample, a shoulder at 1631 cm(-1) in the amide I band gradually appeared from 45 degrees C for Fe SOD and from 50 degrees C for Mn SOD but another shoulder at 1639 cm(-1) appeared from 50 degrees C for Cu/Zn SOD. The peak at 1631 cm(-1) is due to the intermolecular beta-sheet structure, but the peak at 1639 cm(-1) corresponds to the major intramolecular beta-sheet with less random coil structure. This reveals that in the first heating process the transformation from random coil/alpha-helix structure to beta-sheet structure initiated from around 45-50 degrees C. There was about 16-22% compositional change resulting from that transformation. However, both additional shoulders stood there and did not restore to their original spectra even with cooling to room temperature, suggesting the denaturation and irreversible properties of the solid SODs after heating. The thermal-dependent denaturation and irreversibility of Mn SOD, Cu/Zn SOD and Fe SOD were clearly evidenced by the increase in intramolecular and intermolecular beta-sheet structure.     

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.     

Comparative Fourier transform infrared spectroscopy study of cold-, pressure-, and heat-induced unfolding and aggregation of myoglobin

We studied the cold unfolding of myoglobin with Fourier transform infrared spectroscopy and compared it with pressure and heat unfolding. Because protein aggregation is a phenomenon with medical as well as biotechnological implications, we were interested in both the structural changes as well as the aggregation behavior of the respective unfolded states. The cold- and pressure-induced unfolding both yield a partially unfolded state characterized by a persistent amount of secondary structure, in which a stable core of G and H helices is preserved. In this respect the cold- and pressure-unfolded states show a resemblance with an early folding intermediate of myoglobin. In contrast, the heat unfolding results in the formation of the infrared bands typical of intermolecular antiparallel beta-sheet aggregation. This implies a transformation of alpha-helix into intermolecular beta-sheet. H/2H-exchange data suggest that the helices are first unfolded and then form intermolecular beta-sheets. The pressure and cold unfolded states do not give rise to the intermolecular aggregation bands that are typical for the infrared spectra of many heat-unfolded proteins. This suggests that the pathways of the cold and pressure unfolding are substantially different from that of the heat unfolding. After return to ambient conditions the cold- or pressure-treated proteins adopt a partially refolded conformation. This aggregates at a lower temperature (32 degrees C) than the native state (74 degrees C).     

Secondary structure of bovine beta-lactoglobulin B

Secondary-structure-prediction algorithms have been used to find the segments of beta-lactoglobulin sequence most likely to fit the circular dichroism assignment of 15% alpha-helix, 50% beta-sheet, and 15-20% reverse turn. A number of segments may have an alpha-helical conformation but the most prominent region of alpha-helix is from residue 129 to 143. A further probable alpha-helix segment is residues 65-76. The number of residues predicted to occur in segments of beta-sheet structure is less than expected. However, the most likely segments are for residues 1-6, 11-16, 39-45, 80-85, 92-96, 101-107, 117-123, and 145-151. Predicted reverse-turn tetrapeptides are residues 7-10, 49-52, 61-64, 88-91, and 112-115. These predicted secondary structures are consistent with the low-resolution structure of the molecule determined by X-ray diffraction studies.     

Structural dynamic of a self-assembling peptide d-EAK16 made of only D-amino acids

We here report systematic study of structural dynamics of a 16-residue self-assembling peptide d-EAK16 made of only D-amino acids. We compare these results with its chiral counterpart L-form, l-EAK16. Circular dichroism was used to follow the structural dynamics under various temperature and pH conditions. At 25 degrees C the d-EAK16 peptide displayed a typical beta-sheet spectrum. Upon increasing the temperature above 70 degrees C, there was a spectrum shift as the 218 nm valley widens toward 210 nm. Above 80 degrees C, the d-EAK16 peptide transformed into a typical alpha-helix CD spectrum without going through a detectable random-coil intermediate. When increasing the temperature from 4 degrees C to 110 degrees C then cooling back from 110 degrees C to 4 degrees C, there was a hysteresis: the secondary structure from beta-sheet to alpha-helix and then from alpha-helix to beta-sheet occurred. d-EAK16 formed an alpha-helical conformation at pH0.76 and pH12 but formed a beta-sheet at neutral pH. The effects of various pH conditions, ionic strength and denaturing agents were also noted. Since D-form peptides are resistant to natural enzyme degradation, such drastic structural changes may be exploited for fabricating molecular sensors to detect minute environmental changes. This provides insight into the behaviors of self-assembling peptides made of D-amino acids and points the way to designing new peptide materials for biomedical engineering and nanobiotechnology.     

Secondary conformations and temperature effect on structural transformation of amyloid beta (1-28), (1-40) and (1-42) peptides

Secondary structure of three amyloid b-peptides [A beta(1-28), A beta(1-40) and A beta(1-42)] in the solid state was respectively determined by Fourier transform infrared (FT-IR) microspectroscopy. Their thermal-dependent structural transformation were also investigated by FT-IR microspectroscopy equipped with a thermal analyzer. The present result demonstrates that the solid-state A beta(1-28), A beta(1-40) and A beta(1-42) peptides showed a significant IR spectral difference in the amide I and II bands. The secondary conformation of A beta(1-28) peptide was the combination of major beta-sheet and minor alpha-helix with little random coil structures, but A beta(1-40) peptide showed the co-existence of major beta-sheet and minor random coil with little alpha-helix structures. A beta(1-42) peptide mainly consisted of the predominant b-sheet structure. Although the intact A beta(1-28), A beta(1-40) or A beta(1-42) peptide exhibits a different secondary structure, a similar beta-conformation may form after thermal treatment. A thermal-dependent transition was found for solid A beta(1-28) and A beta(1-40) peptides near 40 degrees C and 45 degrees C, respectively. There was no transition temperature for solid A beta(1-42) peptide, however, due to only a very little level of alpha-helix and random coil structure containing in the solid A beta(1-42) peptide. The thermal denaturation plays an important role in the structural transformation from alpha-helix/random coil to beta-sheet.     

Formation of insulin amyloid fibrils followed by FTIR simultaneously with CD and electron microscopy

Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and electron microscopy (EM) have been used simultaneously to follow the temperature-induced formation of amyloid fibrils by bovine insulin at acidic pH. The FTIR and CD data confirm that, before heating, insulin molecules in solution at pH 2.3 have a predominantly native-like alpha-helical structure. On heating to 70 degrees C, partial unfolding occurs and results initially in aggregates that are shown by CD and FT-IR spectra to retain a predominantly helical structure. Following this step, changes in the CD and FTIR spectra occur that are indicative of the extensive conversion of the molecular conformation from alpha-helical to beta-sheet structure. At later stages, EM shows the development of fibrils with well-defined repetitive morphologies including structures with a periodic helical twist of approximately 450 A. The results indicate that formation of fibrils by insulin requires substantial unfolding of the native protein, and that the most highly ordered structures result from a slow evolution of the morphology of the initially formed fibrillar species.     

Spectroscopic studies of fibrinogen and its plasmin-derived fragments.

The secondary structure of human fibrinogen and its plasmin-fragments have been studied by FTIR spectroscopy. The quantitative results for fibrinogen are in good agreement with previous studies using circular dichroism spectroscopy. After treatment of fibrinogen with plasmin in buffer containing Ca2+, two major fragments are produced: fragment E (Mw 45,000) and fragment D (Mw 100,000). Fragment E is shown to contain 50% alpha-helical values, attributed to its coiled-coil portions, and minor beta-strands and turn structures. Its deuteration gives evidence of the presence of solvent-exposed alpha-helical structures. On the other hand, fragment D contains a distribution of secondary structure values of 35% alpha-helix, 29% beta-sheet segments and 17% turn structures. Fragment D itself has two domains: a portion of the original coiled-coil and also a thermally labile globular domain. The coiled-coil portion (Mw 27,000) was isolated and showed a high alpha-helical content (around 70%). The globular domain is estimated to be rich in beta-sheet structures. The spectra of fibrin clots formed in Ca(2+)-containing buffer have a lower amide I/amide II ratio than fibrinogen spectra, which is interpreted as being due to aggregation.     

Effects of electrospinning and solution casting protocols on the secondary structure of a genetically engineered dragline spider silk analogue investigated via Fourier transform Raman spectroscopy

Micrometer and submicrometer diameter fibers of recombinant dragline spider silk analogues, synthesized via protein engineering strategies, have been electrospun from 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and compared with cast films via Raman spectroscopy in order to assess changes in protein conformation that may result from the electrospinning process. Although the solvent casting process was shown to result in predominantly beta-sheet conformation similar to that observed in the bulk, the electrospinning process causes a major change in conformation from beta-sheet to alpha-helix. A possible mechanism involving electric field-induced stabilization of alpha-helical segments in HFIP solution during the electrospinning process is discussed.     

Determination of alpha-helix and beta-sheet stability in the solid state: a solid-state NMR investigation of poly(L-alanine)

The relative stability of alpha-helix and beta-sheet secondary structure in the solid state was investigated using poly(L-alanine) (PLA) as a model system. Protein folding and stability has been well studied in solution, but little is known about solid-state environments, such as the core of a folded protein, where peptide packing interactions are the dominant factor in determining structural stability. (13)C cross-polarization with magic angle spinning (CPMAS) NMR spectroscopy was used to determine the backbone conformation of solid powder samples of 15-kDa and 21.4-kDa PLA before and after various sample treatments. Reprecipitation from helix-inducing solvents traps the alpha-helical conformation of PLA, although the method of reprecipitation also affects the conformational distribution. Grinding converts the secondary structure of PLA to a final steady-state mixture of 55% beta-sheet and 45% alpha-helix at room temperature regardless of the initial secondary structure. Grinding PLA at liquid nitrogen temperatures leads to a similar steady-state mixture with 60% beta-sheet and 40% alpha-helix, indicating that mechanical shear force is sufficient to induce secondary structure interconversion. Cooling the sample in liquid nitrogen or subjecting it to high pressure has no effect on secondary structure. Heating the sample without grinding results in equilibration of secondary structure to 50% alpha-helix/50% beta-sheet at 100 degrees C when starting from a mostly alpha-helical state. No change was observed upon heating a beta-sheet sample, perhaps due to kinetic effects and the different heating rate used in the experiments. These results are consistent with beta-sheet approximately 260 J/mol more stable than alpha-helix in solid-state PLA.     

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.     

Pulmonary surfactant-associated polypeptide C in a mixed organic solvent transforms from a monomeric alpha-helical state into insoluble beta-sheet aggregates.

In the 35-residue pulmonary surfactant-associated lipopolypeptide C (SP-C), the stability of the valyl-rich alpha-helix comprising residues 9-34 has been monitored by circular dichroism, nuclear magnetic resonance, and Fourier transform infrared spectroscopy in both a mixed organic solvent and in phospholipid micelles. The alpha-helical form of SP-C observed in freshly prepared solutions in a mixed solvent of CHCl3/CH3OH/0.1 M HCl 32:64:5 (v/v/v) at 10 degrees C undergoes within a few days an irreversible transformation to an insoluble aggregate that contains beta-sheet secondary structure. Hydrogen exchange experiments revealed that this conformational transition proceeds through a transition state with an Eyring free activation enthalpy of about 100 kJ mol(-1), in which the polypeptide segment 9-27 largely retains a helical conformation. In dodecylphosphocholine micelles, the helical form of SP-C was maintained after seven weeks at 50 degrees C. The alpha-helical form of SP-C thus seems to be the thermodynamically most stable state in this micellar environment, whereas its presence in freshly prepared samples in the aforementioned mixed solvent is due to a high kinetic barrier for unfolding. These observations support a previously proposed pathway for in vivo synthesis of SP-C through proteolytic processing from a 21-kDa precursor protein.     

Conformational studies on a peptide fragment representing the RNA-binding N-terminus of a viral coat protein using circular dichroism and NMR spectroscopy

Conformational studies were performed on a synthetic pentacosapeptide representing the RNA-binding N-terminal region of the coat protein of cowpea chlorotic mottle virus. The effects of ionic strength, addition of (oligo)phosphates and temperature on the conformation of this highly positively charged peptide containing six arginines and three lysines were studied. CD experiments show that the peptide has 15-18% alpha-helical conformation and about 80% random-coil conformation in the absence of inorganic salt at 25 degrees C, and 20-21% alpha-helical conformation under the same conditions at 10 degrees C. Addition of inorganic salts results in an increase of alpha-helix content, up to 42% in the presence of oligophosphate with an average chain length of 18 phosphates, which was used as an RNA analog. NMR experiments show that the alpha-helix formation starts in the region between Thr9 and Gln12, and is extended in the direction of the C terminus. Relaxation measurements show that binding to oligophosphates of increasing length results in reduced internal mobilities of the positively charged side chains of the arginyl and lysyl residues and of the side chain of Thr9 in the alpha-helical region. The alpha-helix formation in the N-terminal part of this viral coat protein upon binding of phosphate groups to the positively charged side chains is suggested to play an essential role in RNA binding.     

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.     

Conformational intermediates in the folding of a coiled-coil model peptide of the N-terminus of tropomyosin and alpha alpha-tropomyosin.

Circular dichroism was used to study the folding of alpha alpha-tropomyosin and AcTM43, a 43-residue peptide designed to serve as a model for the N-terminal domain of tropomyosin. The sequence of the peptide is AcMDAIKKKMQMLKLDVENLLDRLEQLEADLKALEDRYKQLEGGC. The peptide appeared to form a coiled coil at low temperatures (< 25 degrees C) in buffers with physiological ionic strength and pH. The folding and unfolding of the peptide, however, were noncooperative. When CD spectra were examined as a function of temperature, the apparent degree of folding differed when the ellipticity was followed at 222, 208, and 280 nm. Deconvolution of the spectra suggested that at least three component curves contributed to the CD in the far UV. One component curve was similar to the CD spectrum of the coiled-coil alpha-helix of native alpha alpha-tropomyosin. The second curve resembled the spectrum of single-stranded short alpha-helical segments found in globular proteins. The third was similar to that of polypeptides in the random coil conformation. These results suggested that as the peptide folded, the alpha-helical content increased before most of the coiled coil was formed. When the CD spectrum of striated muscle alpha alpha-tropomyosin was examined as a function of temperature, the unfolding was also not totally cooperative. As the temperature was raised from 0 to 25 degrees C, there was a decrease in the coiled coil and an increase in the conventional alpha-helix type spectrum without formation of random coil. The major transition, occurring at 40 degrees C, was a cooperative transition characterized by the loss of all of the remaining coiled coil and a concomitant increase in random coil.     

Effects of oligomerization and secondary structure on the surface behavior of pulmonary surfactant proteins SP-B and SP-C

The relationship among protein oligomerization, secondary structure at the interface, and the interfacial behavior was investigated for spread layers of native pulmonary surfactant associated proteins B and C. SP-B and SP-C were isolated either from butanol or chloroform/methanol lipid extracts that were obtained from sheep lung washings. The proteins were separated from other components by gel exclusion chromatography or by high performance liquid chromatography. SDS gel electrophoresis data indicate that the SP-B samples obtained using different solvents showed different oligomerization states of the protein. The CD and FTIR spectra of SP-B isolated from all extracts were consistent with a secondary structure dominated by alpha-helix. The CD and FTIR spectra of the first SP-C corresponded to an alpha-helical secondary structure and the spectra of the second SP-C corresponded to a mixture of alpha-helical and beta-sheet conformation. In contrast, the spectra of the third SP-C corresponded to antiparallel beta-sheets. The interfacial behavior was characterized by surface pressure/area (pi-A) isotherms. Differences in the oligomerization state of SP-B as well as in the secondary structure of SP-C all produce significant differences in the surface pressure/area isotherms. The molecular cross sections determined from the pi-A isotherms and from dynamic cycling experiments were 6 nm(2)/dimer molecule for SP-B and 1.15 nm(2)/molecule for SP-C in alpha-helical conformation and 1.05 nm(2)/molecule for SP-C in beta-sheet conformation. Both the oligomer ratio of SP-B and the secondary structure of SP-C strongly influence organization and behavior of these proteins in monolayer assemblies. In addition, alpha-helix --> beta-sheet conversion of SP-C occurs simply by an increase of the summary protein/lipid concentration in solution.