Secondary structure and thermostability of the phage P22 tailspike. XX. Analysis by Raman spectroscopy of the wild-type protein and a temperature-sensitive folding mutant

The thermostable tailspike endorhamnosidase of bacteriophage P22 has been investigated by laser Raman spectroscopy to determine the protein's secondary structure and the basis of its thermostability. The conformation of the native tailspike, determined by Raman amide I and amide III band analyses, is 52 to 61% beta-sheet, 24 to 27% alpha-helix, 15 to 21% beta-turn and 0 to 10% other structure types. The secondary structure of the wild-type tailspike, as monitored by the conformation-sensitive Raman amide bands, was stable to 80 degrees C, denatured reversibly between 80 and 90 degrees C, and irreversibly above 90 degrees C. The purified native form of a temperature-sensitive folding mutant (tsU38) contains secondary structures virtually identical to those in the wild-type in aqueous solution at physiological conditions (0.05 M-Na+ (pH 7.5], at both permissive (20 degrees C) and restrictive (40 degrees C) temperatures. This supports previous results showing that the mutational defect at 40 degrees C affects intermediates in the folding pathway rather than the native structure. At temperatures above 60 degrees C the wild-type and mutant forms were distinguishable: the reversible and irreversible denaturation thresholds were approximately 15 to 20 degrees C lower in the mutant than in the wild-type protein. The irreversible denaturation of the mutant tailspikes led to different aggregation/polymerization products from the wild-type, indicating that the mutation altered the unfolding pathway. In both cases only a small percentage of the native secondary structure was altered by irreversible thermal denaturation, indicating that the aggregated states retain considerable native structure.     

Structural and functional relationships in 5'-nucleotidase from bull seminal plasma. A Fourier transform infrared study.

Fourier transform infrared spectroscopy (FTIR) was used to investigate the secondary structure of 5'-nucleotidase from bull seminal plasma (BSP). Spectra of protein in both D2O and H2O were analyzed by deconvolution and second derivative methods in order to observe the overlapping components of the amide I band. The protein, which is made up of two apparently identical subunits and which contains two zinc atoms, was studied in its native form, in the presence of dithiotreitol (DTT) and after removal of the two zinc atoms by means of nitrilotriacetic acid (NTA). Deconvolved and second derivative spectra of amide I band showed that the native protein contains mostly beta-sheet structure with a minor content of alpha-helix. The quantitative analysis of the amide I components was performed by a curve-fitting procedure which revealed 54% beta-sheet, 18% alpha-helix, 22% beta-turns and 6% unordered structure. The second derivative and deconvolved spectra of amide I band showed that no remarkable changes in the secondary structure of 5'-nucleotidase were induced by either DTT or NTA. These results were confirmed by the curve-fitting analysis where little or no changes occurred in the relative content of amide I components when the protein was treated with DTT or with NTA. Major changes, however, were observed in the thermal denaturation behavior of the protein. The native protein showed denaturation at temperatures between 70 and 75 degrees C, while the maximum of denaturation was observed between 65 and 70 degrees C and between 55 and 60 degrees C in the presence of NTA and DTT, respectively. The results obtained indicate that the two separate subunits of the protein have essentially the same secondary structure as that of the native enzyme.     

Methionine adenosyltransferase alpha-helix structure unfolds at lower temperatures than beta-sheet: a 2D-IR study

Two-dimensional infrared spectroscopy has been used to characterize rat liver methionine adenosyltransferase and the events taking place during its thermal unfolding. Secondary structure data have been obtained for the native recombinant enzyme by fitting the amide I band of infrared spectra. Thermal denaturation studies allow the identification of events associated with individual secondary-structure elements during temperature-induced unfolding. They are correlated to the changes observed in enzyme activity and intrinsic fluorescence. In all cases, thermal denaturation proved to be an irreversible process, with a T(m) of 47-51 degrees C. Thermal profiles and two-dimensional infrared spectroscopy show that unfolding starts with alpha-helical segments and turns, located in the outer part of the protein, whereas extended structure, associated with subunit contacts, unfolds at higher temperatures. The data indicate a good correlation between the denaturation profiles obtained from activity measurements, fluorescence spectroscopy, and the behavior of the infrared bands. A study of the sequence of events that takes place is discussed in light of the previous knowledge on methionine adenosyltransferase structure and oligomerization pathway.     

Fourier transform infrared spectroscopy suggests unfolding of loop structures precedes complete unfolding of pig citrate synthase

Pig citrate synthase (PCS) can be used as a model enzyme to gain some insight into the structural basis of protein thermostability. The thermal unfolding characteristics of the specific secondary structure elements within PCS were monitored in detail by following changes in its amide I band components. The result of our study indicates that PCS undergoes irreversible thermal denaturation. Detailed analysis reveals that the different secondary structures display a multistep transition with a major and a minor transition at different temperatures and a very small initial transition at the same temperature (30 degrees C). A plot of temperature-induced changes in (1)H-(2)H exchange, the decrease in the absorbance of the alpha-helical structures, and the increase in the absorbance of aggregated structures all have in common a multistep transition, the minor one centered at 45 degrees C and the major one around 59 degrees C. In contrast, a band that is tentatively assigned to loop structures displays these same minor and major transitions but at lower temperatures (39 and 52 degrees C, respectively). The transition, which occurs at 39-45 degrees C, is not associated with the appearance of aggregated structures. This transition may reflect a change in the tertiary structure of the protein. However, the final transition, which occurs at a higher temperature (52-59 degrees C), reflects unfolding and aggregation of the polypeptide chains. The Fourier transform infrared (FTIR) analysis suggests that PCS has a thermolabile region that unfolds first, some 7 degrees C below the main unfolding of the protein. We propose that this reflects the unfolding of the highly flexible loop segments, which in turn triggers the unfolding of the predominantly helical core structure of PCS.     

Structural and conformational changes of beta-lactoglobulin B: an infrared spectroscopic study of the effect of pH and temperature

Infrared spectra of 2.5 mM solutions of beta-lactoglobulin B were recorded as a function of pH (from pH 2 to pH 13) and as a function of temperature (from -100 degrees C to +90 degrees C). An analysis of the pH- and temperature-induced changes in the secondary structure was performed based on changes in the conformation-sensitive amide I bands of beta-lactoglobulin. Whereas the total amount of beta-structure remains constant (56-59%) between pH 2 and pH 10, the proportions of the various beta-components do change. In particular, the dimerization of the monomeric protein, induced by raising the pH from 2 to 3 , leads to an increase in the intensity of the 1636 cm-1 band (associated with antiparallel beta-sheet), at the expense of the 1626 cm-1 band (associated with exposed beta-strands). Both the thermal and alkaline denaturation of beta-lactoglobulin occur in two distinct stages. Although the spectra (i.e., the structures) after complete thermal or alkaline denaturation are clearly different, the spectrum of the protein after the first stage of thermal denaturation (at about 60 degrees C) is the same as that after the first stage of alkaline denaturation (at pH 11), suggesting a common denaturation intermediate, which probably represents a crossover point in a complex potential hypersurface.     

The isolated C-terminal (F2) fragment of the Escherichia coli tryptophan synthase beta 2-subunit folds into a stable, organized nonnative conformation

Proteolysis of the beta 2-subunit of Escherichia coli tryptophan synthase by the endoproteinase Glu C from Staphylococcus aureus V8 yields a peptide, F2, corresponding to the C-terminal 101 residues of the beta-chain. The conformation and stability of isolated F2 in phosphate buffer at pH 7.8 (where native beta 2 is stable) have been investigated. Circular dichroism spectra in the far-UV showed the presence of large amounts of secondary structure (19% alpha-helices, 34% extended beta-structures). Circular dichroism spectra in the near-UV and sedimentation velocity studies indicated an open globular structure with the aromatic side chains in a symmetric (or disordered) environment. NMR spectra and rates of amide proton exchange showed that F2 fluctuates rapidly between several conformations. The thermal denaturation of F2 observed by the loss of far-UV circular dichroism with increasing temperature appeared noncooperative, and indicates a high thermal stability (Tm = 70 degrees C). Differential scanning microcalorimetry confirmed the absence of cooperativity and indicated a very low value for the calorimetric enthalpy of denaturation (delta H = 17 kJ/mol). All these properties were compatible with a molten globule. However, the low sedimentation coefficient of F2 suggested a very hydrated and/or expanded structure, and the secondary structure content of isolated F2 (see above) differed widely from that reported in the literature for F2 within the context of native beta 2 (49% alpha-helices and 13% extended beta-structures). Thus, neither the secondary nor the tertiary structure of isolated F2 resembled those of native F2. In this respect, isolated F2 is not a "molten globule".(ABSTRACT TRUNCATED AT 250 WORDS)     

A Fourier transform infrared investigation of the structural differences between ribonuclease A and ribonuclease S

Fourier transform infrared spectroscopy was used to investigate the small conformational differences which exist between ribonuclease A and ribonuclease S in aqueous systems. Deconvolution and derivative methods were used to observe the overlapping components of the amide I and II bands. These proteins give identical spectra in H2O and after complete exchange in 2H2O. However structural differences are revealed by monitoring the rate of 1H-2H exchange by Fourier transform infrared spectroscopy. At equivalent times of exposure in 2H2O buffer ribonuclease S undergoes greater isotopic exchange than ribonuclease A. Thus complete exchange takes place for ribonuclease S but not ribonuclease A after incubation at room temperature for 8 days. Complete 1H-2H exchange of ribonuclease A was achieved by incubation at 62 degrees C for 30 min. The available X-ray data and comparison with the infrared spectra of other soluble proteins was used to assign the components of the amide I and II bands to various secondary structures. In particular, band shifts observed during the later stages of exchange are associated with slowly exchanging residues in beta-strand and alpha-helical regions. The higher rate of exchange for ribonuclease S is associated with a greater conformational flexibility and a more open structure. The results show that it is necessary to be cautious in making band assignments based on exchange methods unless the extent of exchange is known. Furthermore, it is seen that the combination of Fourier transform infrared spectroscopy and hydrogen-deuterium exchange is a powerful technique for revealing small differences in protein secondary structure.     

The role of the native lipids and lattice structure in bacteriorhodopsin protein conformation and stability as studied by temperature-dependent Fourier transform-infrared spectroscopy

We report the effect of partial delipidation and monomerization on the protein conformational changes of bacteriorhodopsin (bR) as a function of temperature. Removal of up to 75% of the lipids is known to have the lattice structure of the purple membrane, albeit as a smaller unit cell, whereas treatment by Triton monomerizes bR into micelles. The effects of these modifications on the protein secondary structure is analyzed by monitoring the protein amide I and amide II bands in the Fourier transform-infrared (FT-IR) spectra. It is found that removal of the first 75% of the lipids has only a slight effect on the secondary structure at physiological temperature, whereas monomerizing bR into micelles alters the secondary structure considerably. Upon heating, the bR monomer is found to have a very low thermal stability compared with the native bR with its melting point reduced from 97 to 65 degrees C, and the pre-melting transition in which the protein changes conformation in native bR at 80 degrees C could not be observed. Also, the N[bond]H to N[bond]D exchange of the amide II band is effectively complete at room temperature, suggesting that there are no hydrophobic regions that are protected from the aqueous medium, possibly explaining the low thermal stability of the monomer. On the other hand, 75% delipidated bR has its melting temperature close to that of the native bR and does have a pre-melting transition, although the pre-melting transition occurs at significantly higher temperature than that of the native bR (91 degrees C compared with 80 degrees C) and is still reversible. Furthermore, we have also observed that the reversibility of this pre-melting transition of both native and partially delipidated bR is time-dependent and becomes irreversible upon holding at 91 degrees C between 10 and 30 min. These results are discussed in terms of the lipid and lattice contribution to the protein thermal stability of native bR.     

Fourier transform infrared evidence for a predominantly alpha-helical structure of the membrane bound channel forming COOH-terminal peptide of colicin E1.

The structure of the membrane bound state of the 178-residue thermolytic COOH-terminal channel forming peptide of colicin E1 was studied by polarized Fourier transform infrared (FTIR) spectroscopy. This fragment was reconstituted into DMPC liposomes at varying peptide/lipid ratios ranging from 1/25-1/500. The amide I band frequency of the protein indicated a dominant alpha-helical secondary structure with limited beta- and random structures. The amide I and II frequencies are at 1,656 and 1,546 cm-1, close to the frequency of the amide I and II bands of rhodopsin, bacteriorhodopsin and other alpha-helical proteins. Polarized FTIR of oriented membranes revealed that the alpha-helices have an average orientation less than the magic angle, 54.6 degrees, relative to the membrane normal. Almost all of the peptide groups in the membrane-bound channel protein undergo rapid hydrogen/deuterium (H/D) exchange. These results are contrasted to the alpha-helical membrane proteins, bacteriorhodopsin, and rhodopsin.     

FTIR study on heat-induced and pressure-assisted cold-induced changes in structure of bovine alpha-lactalbumin: stabilizing role of calcium ion

The second derivative FTIR study of heat-induced and pressure-assisted cold-induced changes in the secondary structure of bovine alpha-lactalbumin was carried out for native holoprotein and calcium ion depleted apoprotein. The secondary structure and compactness of alpha-lactalbumin were examined in a temperature range from 20 to 80 degrees C during the heat treatment and 20 to -15 degrees C during the pressure-assisted cold treatment. This was the first FTIR study on the pressure-assisted cold denaturation of a protein. Because protein solutions had close to neutral pD and low ionic strength, the apoprotein remained in the molten globule state and the holoform maintained its native tertiary structure. In order to distinguish between unfolding-related and partially deuterated exchange-related spectral changes, we examined both the fully deuterated holoform and the partially deuterated holoform. The quantitative analysis of the spectral changes in the amide I/I' vibrational band revealed that the 3(10) helices were more prone to thermal unfolding than the alpha helices. We observed that the protein's compactness and secondary structure were both considerably stabilized against an increase and decrease in temperature by the presence of a calcium ion. Under the conditions of this study, only the apoprotein was susceptible to the cold denaturation. In contrast to this, an unexpected linear increase of the alpha-helical content was observed upon the cooling of the holoprotein under high pressure. The results were discussed in reference to the existing crystallographic data for crystals of human alpha-lactalbumin grown at two different temperatures.     

Topology of sarcoplasmic reticulum Ca2+-ATPase: an infrared study of thermal denaturation and limited proteolysis.

Sarcoplasmic reticulum Ca2+-ATPase structure and organization in the membrane has been studied by infrared spectroscopy by decomposition of the amide I band. Besides the component bands assignable to secondary structure elements such as alpha-helix, beta-sheet, etc...., two unusual bands, one at 1,645 cm(-1) in H2O buffer and the other at 1,625 cm(-1) in D2O buffer are present. By perturbing the protein using temperature and limited proteolysis, the band at 1,645 cm(-1) is tentatively assigned to alpha-helical segments located in the cytoplasmic domain and coupled to beta-sheet structure, whereas the band at 1,625 cm(-1) arises probably from monomer-monomer contacts in the native oligomeric protein. The secondary structure obtained is 33% alpha-helical segments in the transmembrane plus stalk domain; 20% alpha-helix and 22% beta-sheet in the cytoplasmic domain plus 19% turns and 6% unordered structure. Thermal unfolding of Ca2+-ATPase is a complex process that cannot be described as a two-state denaturation. The results obtained are compatible with the idea that the protein is an oligomer at room temperature. The loss of the 1,625 cm(-1) band upon heating would be consistent with a disruption of the oligomers in a process that later gives rise to aggregates (appearance of the 1,618 cm(-1) band). This picture would also be compatible with early results suggesting that processes governing Ca2+ accumulation and ATPase activity are uncoupled at temperatures above 37 degrees C, so that while ATPase activity proceeds at high rates, Ca2+ accumulation is inhibited.     

Thermal stability and conformational transitions of scrapie amyloid (prion) protein correlate with infectivity.

The scrapie amyloid (prion) protein (PrP27-30) is the protease-resistant core of a larger precursor (PrPSc) and a component of the infectious scrapie agent; the potential to form amyloid is a result of posttranslational event or conformational abnormality. The conformation, heat stability, and solvent-induced conformational transitions of PrP27-30 were studied in the solid state in films by CD spectroscopy and correlated with the infectivity of rehydrated and equilibrated films. The exposure of PrP27-30 in films to 60 degrees C, 100 degrees C, and 132 degrees C for 30 min did not change the beta-sheet secondary structure; the infectivity slightly diminished at 132 degrees C and correlated with a decreased solubility of PrP27-30 in sodium dodecyl sulfate (SDS), probably due to cross-linking. Exposing PrP27-30 films to formic acid (FA), trifluoroacetic acid (TFA), trifluoroethanol (TFE), hexafluoro-2-propanol (HFIP), and SDS transformed the amide CD band, diminished the mean residue ellipticity of aromatic bands, and inactivated scrapie infectivity. The convex constraint algorithm (CAA) deconvolution of the CD spectra of the solvent-exposed and rehydrated solid state PrP27-30 identified five common spectral components. The loss of infectivity quantitatively correlated with a decreasing proportion of native, beta-pleated sheet-like secondary structure component, an increasing amount of alpha-helical component, and an increasingly disordered tertiary structure. The results demonstrate the unusual thermal stability of the beta-sheet secondary structure of PrP27-30 protein in the solid state. The conformational perturbations of PrP27-30 parallel the changes in infectivity and suggest that the beta-sheet structure plays a key role in the physical stability of scrapie amyloid and in the ability to propagate and replicate scrapie.     

Secondary structure components and properties of the melibiose permease from Escherichia coli: a fourier transform infrared spectroscopy analysis

The structure of the melibiose permease from Escherichia coli has been investigated by Fourier transform infrared spectroscopy, using the purified transporter either in the solubilized state or reconstituted in E. coli lipids. In both instances, the spectra suggest that the permease secondary structure is dominated by alpha-helical components (up to 50%) and contains beta-structure (20%) and additional components assigned to turns, 3(10) helix, and nonordered structures (30%). Two distinct and strong absorption bands are recorded at 1660 and 1653 cm(-1), i.e., in the usual range of absorption of helices of membrane proteins. Moreover, conditions that preserve the transporter functionality (reconstitution in liposomes or solubilization with dodecyl maltoside) make possible the detection of two separate alpha-helical bands of comparable intensity. In contrast, a single intense band, centered at approximately 1656 cm(-1), is recorded from the inactive permease in Triton X-100, or a merged and broader signal is recorded after the solubilized protein is heated in dodecyl maltoside. It is suggested that in the functional permease, distinct signals at 1660 and 1653 cm(-1) arise from two different populations of alpha-helical domains. Furthermore, the sodium- and/or melibiose-induced changes in amide I line shape, and in particular, in the relative amplitudes of the 1660 and 1653 cm(-1) bands, indicate that the secondary structure is modified during the early step of sugar transport. Finally, the observation that approximately 80% of the backbone amide protons can be exchanged suggests high conformational flexibility and/or a large accessibility of the membrane domains to the aqueous solvent.     

Thermodynamics of single peptide bond cleavage in bovine pancreatic trypsin inhibitor (BPTI)

A major goal of this paper was to estimate a dynamic range of equilibrium constant for the opening of a single peptide bond in a model protein, bovine pancreatic trypsin inhibitor (BPTI). Ten mutants of BPTI containing a single Xaa-->Met substitution introduced in different parts of the molecule were expressed in Escherichia coli. The mutants were folded, purified to homogeneity, and cleaved with cyanogen bromide to respective cleaved forms. Conformation of the intact mutants was similar to the wildtype, as judged from their circular dichroism spectra. Substantial conformational changes were observed on the chemical cleavage of three single peptide bonds--Met46-Ser, Met49-Cys, and Met53-Thr--located within the C-terminal helix. Cleavage of those peptide bonds caused a significant destabilization of the molecule, with a drop of the denaturation temperature by 56.4 degrees C to 68 degrees C at pH 4.3. Opening of the remaining seven peptide bonds was related to a 10.8 degrees C to 39.4 degrees C decrease in T(den). Free energies of the opening of 10 single peptide bonds in native mutants (Delta G(op,N)) were estimated from the thermodynamic cycle that links denaturation and cleavage free energies. To calculate those values, we assumed that the free energy of opening of a single peptide bond in the denatured state (Delta G(op,D)) was equal to -2.7 kcal/mole, as reported previously. Calculated Delta G(op,N) values in BPTI were in the range from 0.2 to 10 kcal/mole, which was equivalent to a >1 million-fold difference in equilibrium constants. The values of Delta G(op,N) were the largest for peptide bonds located in the C-terminal helix and significantly lower for peptide bonds in the beta-structure or loop regions. It appears that opening constants for single peptide bonds in various proteins span across 33 orders of magnitude. Typical equilibrium values for a single peptide bond opening in a protein containing secondary structure elements fall into negligibly low values, from 10(-3) to 10(-8), and are efficient to ensure stability against proteolysis.     

Structural description of acid-denatured cytochrome c by hydrogen exchange and 2D NMR

Hydrogen exchange and two-dimensional nuclear magnetic resonance (2D NMR) techniques were used to characterize the structure of oxidized horse cytochrome c at acid pH and high ionic strength. Under these conditions, cytochrome c is known to assume a globular conformation (A state) with properties resembling those of the molten globule state described for other proteins. In order to measure the rate of hydrogen-deuterium exchange for individual backbone amide protons in the A state, samples of oxidized cytochrome c were incubated at 20 degrees C in D2O buffer (pD 2.2, 1.5 M NaCl) for time periods ranging from 2 min to 500 h. The exchange reaction was then quenched by transferring the protein to native conditions (pD 5.3). The extent of exchange for 44 amide protons trapped in the refolded protein was measured by 2D NMR spectroscopy. The results show that this approach can provide detailed information on H-bonded secondary and tertiary structure in partially folded equilibrium forms of a protein. All of the slowly exchanging amide protons in the three major helices of native cytochrome c are strongly protected from exchange at acid pH, indicating that the A state contains native-like elements of helical secondary structure. By contrast, a number of amide protons involved in irregular tertiary H-bonds of the native structure (Gly37, Arg38, Gln42, Ile57, Lys79, and Met80) are only marginally protected in the A state, indicating that these H-bonds are unstable or absent. The H-exchange results suggest that the major helices of cytochrome c and their common hydrophobic domain are largely preserved in the globular acidic form while the loop region of the native structure is flexible and partly disordered.     

Interaction of alcohols with serum LDL An infrared study

The interaction of low molecular weight alcohols with low density lipoprotein (LDL) has been studied using amide I band-fitting, thermal profiling and two-dimensional infrared correlation spectroscopy (2D-IR). At 0.3 M alcohol, no changes in secondary structure are observed. In the presence of 1 M alcohol, ethanol and propanol decreases protein denaturation temperature and produces changes in the amide I thermal profiles of protein components and in the lipid bands. The 2D-IR synchronous map corresponding to protein or lipid component at 20-37 degrees C suggests differences in the presence of propanol. The asynchronous map corresponding to the lipid component indicates changes in bandwidth, compatible with a more fluid environment. In the 37-80 degrees C temperature range the thermal profile is different in the presence of propanol, both for the lipid and protein components. The results presented show that when alcohols affect the protein component, the lipid spectrum also varies pointing to an effect on the lipid-protein interaction.     

Thermal denaturation of globular proteins. Fourier transform-infrared studies of the amide III spectral region.

Fourier transform-infrared (FT-IR) spectra are reported for the amide III spectral region of the native and thermally denatured forms of chymotrypsinogen, ribonuclease, bovine serum albumin, and lysozyme. Chymotrypsinogen denatures into structures containing substantial contributions from beta-sheets, while lysozyme and bovine serum albumin show increased amounts of random-coil forms. The changes observed for ribonuclease are quite small. Bovine serum albumin shows at least six bands in the 1,260-1,320 cm-1 region which undergo large intensity changes upon thermal denaturation, and hence are assignable to alpha-helical amide III modes. The large number of observed bands suggests that slight variations in helical geometry, symmetry, or interactions result in changed amide III frequencies, so that simple correlations between narrow frequency ranges and secondary structures may not be applicable for this mode. A widened frequency range is suggested as diagnostic for helical structures.     

Protein secondary structure of the isolated photosystem II reaction center and conformational changes studied by Fourier transform infrared spectroscopy.

The secondary structure of the photosystem II (PSII) reaction center isolated from pea chloroplasts has been characterized by Fourier transform infrared (FTIR) spectroscopy. Spectra were recorded in aqueous buffers containing H2O or D2O; the detergent present for most measurements was dodecyl maltoside. The broad amide I and amide II bands were analyzed by using second-derivative and deconvolution procedures. Absorption bands were assigned to the presence of alpha-helices, beta-sheets, turns, or random structure. Quantitative analysis revealed that this complex contained a high proportion of alpha-helices (67%) and some antiparallel beta-sheets (9%) and turns (11%). An irreversible decrease in the intensity of the band associated with the alpha-helices occurs upon exposure of the isolated PSII reaction center to bright illumination. This loss of alpha-helical content gave rise to an increase in other secondary structures, particularly beta-sheets. After similar pretreatment with light, sodium dodecyl sulfate polyacrylamide gel electrophoresis reveals lower mobility and solubility of constituent D1 and D2 polypeptides of the PSII reaction center. Some degradation of these polypeptides also occurs. In contrast, there is no change in the mobility of the two subunits of cytochrome b559. In the absence of illumination, the PSII reaction center exchanged into dodecyl maltoside shows good thermal stability as compared with samples in Triton X-100. Only at a temperature of about 60 degrees C do spectral changes take place that are indicative of denaturation.     

Effect of calcium and phosphatidic acid binding on the C2 domain of PKC alpha as studied by Fourier transform infrared spectroscopy.

Fourier transform infrared (FTIR) spectroscopy was used to investigate the structural and thermal denaturation of the C2 domain of PKC alpha (PKC-C2) and its complexes with Ca(2+) and phosphatidic acid vesicles. The amide I regions in the original spectra of PKC-C2 in the Ca(2+)-free and Ca(2+)-bound states are both consistent with a predominantly beta-sheet secondary structure below the denaturation temperatures. Spectroscopic studies of the thermal denaturation revealed that for the PKC-C2 domain alone the secondary structure abruptly changed at 50 degrees C. While in the presence of 2 and 12.5 mM Ca(2+), the thermal stability of the protein increased to 60 and 70 degrees C, respectively. Further studies using a mutant lacking two important amino acids involved in Ca(2+) binding (PKC-C2D246/248N) demonstrated that these mutations were inherently more stable to thermal denaturation than the wild-type protein. Phosphatidic acid binding to the PKC-C2 domain was characterized, and the lipid-protein binding became Ca(2+)-independent when 100 mol% phosphatidic acid vesicles were used. The mutant lacking two Ca(2+) binding sites was also able to bind to phosphatidic acid vesicles. The effect of lipid binding on secondary structure and thermal stability was also studied. Beta-sheet was the predominant structure observed in the lipid-bound state, although the percentage represented by this structure in the total area of the amide I band significantly decreased from 60% in the lipid-free state to 47% in the lipid-bound state. This decrease in the beta-sheet component of the lipid-bound complex correlates well with the significant increase observed in the 1644 cm(-1) band which can be assigned to loops and disordered structure. Thermal stability after lipid binding was very high, and no sign of thermal denaturation was observed in the presence of lipids under the conditions that were studied.     

Fourier transform infrared spectroscopy and site-directed isotope labeling as a probe of local secondary structure in the transmembrane domain of phospholamban.

Phospholamban is a 52-amino acid residue membrane protein that regulates Ca(2+)-ATPase activity in the sarcoplasmic reticulum of cardiac muscle cells. The hydrophobic C-terminal 28 amino acid fragment of phospholamban (hPLB) anchors the protein in the membrane and may form part of a Ca(2+)-selective ion channel. We have used polarized attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy along with site-directed isotope labeling to probe the local structure of hPLB. The frequency and dichroism of the amide I and II bands appearing at 1658 cm-1 and 1544 cm-1, respectively, show that dehydrated and hydrated hPLB reconstituted into dimyristoylphosphatidycholine bilayer membranes is predominantly alpha-helical and has a net transmembrane orientation. Specific local secondary structure of hPLB was probed by incorporating 13C at two positions in the protein backbone. A small band seen near 1614 cm-1 is assigned to the amide I mode of the 13C-labeled amide carbonyl group(s). The frequency and dichroism of this band indicate that residues 39 and 46 are alpha-helical, with an axial orientation that is approximately 30 degrees relative to the membrane normal. Upon exposure to 2H2O (D2O), 30% of the peptide amide groups in hPLB undergo a slow deuterium/hydrogen exchange. The remainder of the protein, including the peptide groups of Leu-39 and Leu-42, appear inaccessible to exchange, indicating that most of the hPLB fragment is embedded in the lipid bilayer. By extending spectroscopic characterization of PLB to include hydrated, deuterated as well as site-directed isotope-labeled hPLB films, our results strongly support models of PLB that predict the existence of an alpha-helical hydrophobic region spanning the membrane domain.