Oriented secondary structure in integral membrane proteins. I. Circular dichroism and infrared spectroscopy of cytochrome oxidase in multilamellar films.

The circular dichroism (CD) of cytochrome oxidase in solution indicates the presence of both alpha-helix (approximately 37%) and B-sheet (approximately 18%). In oriented films generated by the isopotential spin-dry method, the CD measured normal to the film shows a marked decrease in the negative bands at 222 and 208 nm, and a decrease and red shift in the positive band near 195 nm, relative to solution spectra. These features are characteristic of alpha-helices oriented with their helix axes along the direction of light propagation. A quantitative estimate of the orientation, based on the ratio of the rotational strengths of the 208-nm band in the film and in solution, leads to an average angle between the helix axis and the normal to the film, phi alpha of approximately 39 degrees. A method for analyzing infrared (IR) linear dichroism is developed that can be applied to proteins with comparable amounts of alpha-helix and beta-sheet. From analysis of the amide I band, phi alpha is found to lie between 20 and 36 degrees, depending on the angle that the amide I transition moment forms with the helix axis. A survey of the literature on the amide I transition moment direction indicates that a value of approximately 27 degrees is appropriate for standard alpha-helical systems, such as those in cytochrome oxidase. A larger value, near 40 degrees, is reasonable for systems that have distorted alpha-helices, as evidenced by amide I frequencies above 1,660 cm-1, as is the case of bacteriorhodopsin. This conclusion supports phi alpha approximately 36 degrees from IR linear dichroism, in agreement with the CD results. Linear dichroism in the amide I and amide II region indicates that the beta-sheet in cytochrome oxidase is oriented with the carbonyl groups nearly parallel to the plane of the membrane and the chain direction inclined at approximately 40 degrees to the normal. Comparison of these results with tentative identification of transmembrane helices from sequence data suggests that either some of the transmembrane helices are inclined at an unexpectedly large angle to the normal, or the number of such helices has been overestimated. Some putative transmembrane helices may be beta-strands spanning the membrane.     

Nature of forces stabilizing the transmembrane protein bacteriorhodopsin in purple membrane

Analysis of the far-ultraviolet solution and the oriented-film circular dichroic (CD) spectra of the purple membrane (PM) has indicated that the α-helical segments of its sole protein bacteriorhodopsin (bR) can undergo a significant tilting from the normal to the membrane plane during light-dependent hydroxylamine-mediated bleaching of the bR. However, this drastic change in tertiary structure is free of any observable secondary structural changes. This phenomenon can provide an excellent means for studying the relative contributions of forces responsible for the stability of this transmembrane protein within the membrane bilayer. Perturbation of the PM by varying degrees of papain digestion (resulting in changes in the bR ranging from only an elimination of the long COOH-terminal tail to the additional eliminations of the short NH₂-terminal tail and a number of linkage amino acids between the helical segments of the bR) and by chemical cross-linking with dimethyl adipimidate (resulting primarily in the formation of intramolecular cross-links) resulted in a significant increase in this bleaching-induced tilting in all cases except the one in which only the COOH-tail was eliminated. The most severe perturbation (2-wk papain digestion) increased the net tilt angle per segment from 24 to 39° with no indication of any secondary structural changes. Although these perturbations drastically reduced the structural stability of the bR to bleaching, they caused virtually no observable changes in the intramolecular structure of the bR or the supramolecular structure of the PM based on analysis of extensive absorption, linear dichroic, and CD spectra. In addition, study of the bleaching rates for the perturbed PM samples indicated that a linear correlation exists between the calculated initial bleaching rates and the net tilt angles.

Considering the forces generally assumed to account for the stability of transmembrane proteins in membranes, (a) intersegmental hydrogen bonding and electrostatic interactions, (b) electrostatic interactions between hydrophilic polypeptide segments extending outside the bilayer and the many charged lipid heads of the bilayer, and (c) hydrophobic interactions, it is clear that the results of the bleaching experiments eliminate all but perhaps the last as contributing significantly to the bR stability in the PM. Furthermore, they provide more compelling evidence than previously available that the bR is capable of undergoing relatively large retinyldiene-controlled tertiary structural changes and that the chromophoric retinal serves as the most important factor in the native bR structural stability. This dynamic view of the bR bears directly on models proposed for bR function, favoring those in which protein structural metastability, rather than rigidity, is an essential factor. The proteinquake or deformation wave model proposed by this laboratory falls into this category.

     

Structural and orientational constraints of bacteriorhodopsin in purple membranes determined by oriented-sample solid-state NMR spectroscopy

We report for the first time, oriented-sample solid-state NMR experiments, specifically polarization inversion spin exchange at the magic angle (PISEMA) and 1H-15N heteronuclear chemical shift correlation (HETCOR), applied to an integral seven-transmembrane protein, bacteriorhodopsin (bR), in natural membranes. The spectra of [15N]Met-bR revealed clearly distinguishable signals from the helical and loop regions. By deconvolution of the helix resonances, it was possible to establish constraints for some helix tilt angles. It was estimated that the extracellular section of helix B has a tilt of less than 5 degrees from the membrane normal, while the tilt of helix A was estimated to be 18-22 degrees , both of which are in agreement with most crystal structures. Comparison of the experimental PISEMA spectrum with simulated spectra based on crystal structures showed that PISEMA and HETCOR experiments are extremely sensitive to the polytopic protein structure, and the solid-state NMR spectra for membrane-embedded bR matched most favorably with the recent 1FBB electron crystallography structure. These results suggest that this approach has the potential to yield structural and orientational constraints for large integral polytopic proteins whilst intercalated and functionally competent in a natural membrane.     

Polarization-modulated FTIR spectroscopy of lipid/gramicidin monolayers at the air/water interface

Monolayers of gramicidin A, pure and in mixtures with dimyristoylphosphatidylcholine (DMPC), were studied in situ at the air/H2O and air/D2O interfaces by polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). Simulations of the entire set of amide I absorption modes were also performed, using complete parameter sets for different conformations based on published normal mode calculations. The structure of gramicidin A in the DMPC monolayer could clearly be assigned to a beta6.3 helix. Quantitative analysis of the amide I bands revealed that film pressures of up to 25-30 mN/m the helix tilt angle from the vertical in the pure gramicidin A layer exceeded 60 degrees. A marked dependence of the peptide orientation on the applied surface pressure was observed for the mixed lipid-peptide monolayers. At low pressure the helix lay flat on the surface, whereas at high pressures the helix was oriented almost parallel to the surface normal.     

Conformational changes in bacteriorhodopsin studied by infrared attenuated total reflection.

We report on a new method based on Fourier transform infrared (FTIR)-difference spectroscopy for studying the conformational changes occurring during the photocycle of bacteriorhodopsin. Previous studies have been made by measuring the absorbance of an infrared (IR) beam transmitted through a thin hydrated purple membrane film. In contrast, the present study utilizes the technique of attenuated total reflection (ATR). Purple membrane is fixed on the surface of a germanium internal reflection crystal and immersed in a buffer whose pH and ionic composition can be varied. Measurements of the amide I and II absorbance with light polarized parallel and at 45 degrees to the crystal surface reveals that the membrane is highly oriented. An ATR-FTIR-difference spectrum of the light to dark (bR570 to bR548) transition is similar but not identical to the transmittance FTIR-difference spectrum. This disagreement between the two methods is shown to be due in the ATR case to the absorption of transition moments oriented predominantly out of the membrane plane. Raising the pH of La3+ substituted purple membrane films from 6.8 to 8.0 slows the M-decay rate sufficiently so that a bR570 to M412 difference spectrum can be obtained with steady state illumination at room temperature. A comparison of this difference spectrum with that obtained at -23 degrees C using the transmittance method reveals several changes that cannot be attributed to out-of-plane transition moments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Topology and immersion depth of an integral membrane protein by paramagnetic rates from dissolved oxygen

In studies of membrane proteins, knowledge of protein topology can provide useful insight into both structure and function. In this work, we present a solution NMR method for the measurement the tilt angle and average immersion depth of alpha helices in membrane proteins, from analysis of the paramagnetic relaxation rate enhancements arising from dissolved oxygen. No modification to the micelle or protein is necessary, and the topology of both transmembrane and amphipathic helices are readily determined. We apply this method to the measure the topology of a monomeric mutant of phospholamban (AFA-PLN), a 52-residue membrane protein containing both an amphipathic and a transmembrane alpha helix. In dodecylphosphocholine micelles, the amphipathic helix of AFA-PLN was found to have a tilt angle of 87° ± 1° and an average immersion depth of 13.2 ?. The transmembrane helix was found to have an average immersion depth of 5.4 ?, indicating residues 41 and 42 are closest to the micelle centre. The resolution of paramagnetic relaxation rate enhancements from dissolved oxygen compares favourably to those from Ni (II), a hydrophilic paramagnetic species.     

Comparative study of myelin proteolipid apoprotein solvation by multilayer membranes of synthetic DPPC and biological lipid extract from bovine brain. An FT-IR investigation

The interaction between the aqueous form of the myelin proteolipid apoprotein (PLA) and model membranes prepared with either synthetic dipalmitoylphosphatidyl choline (DPPC) or biological lipids extracted from bovine brain (BE) has been investigated by Fourier-Transform IR spectroscopy. IR spectra obtained with lyophilized samples of PLA demonstrated 2 main peaks (amide I and amide II) culminating at 1656 cm-1 and 1545 cm-1, which we assigned to helical conformation. When PLA was solvated in DPPC or BE membranes, both the amide I and amide II features remained located at 1655 cm-1 and 1545 cm-1, although their half-width significantly decreased, demonstrating that the lipid environment favoured alpha helix structures. However differences between both mixtures were detected by measuring the amide I and amide II half-widths as a function of the L:P molar ratio. Moreover, analysis of the 1545/1515 peak intensity ratio brought evidence of different localization and/or molecular arrangement of the protein segments containing tyrosine residues, depending on the lipid composition of the membrane. According to previously published models, these data suggest that recombinants prepared with PLA and BE multilayers better mimic the biological membrane than do DPPC-PLA mixtures.     

Orientation of retinal in purple membrane determined by polarized Raman spectroscopy.

The orientation of the retinal molecule in the purple membrane was determined by polarized Raman spectroscopy for stacked purple membranes. The depolarization ratios of C = C stretching vibration mode were measured for three scattering geometries of purple membrane films. From the depolarization ratios we estimated the tilt angle of the transition dipole moment of retinal to the membrane normal and the rotational angle of the molecular plane along the transition dipole moment of retinal. The molecular plane of M intermediate was found to be almost perpendicular to the membrane plane. We confirmed that the tilt angle was 65 +/- 2 degrees for both bR and M intermediates.     

Infrared dichroism from the X-ray structure of bacteriorhodopsin

A detailed comparison with the three-dimensional protein structure provides a stringent test of the models and parameters commonly used in determining the orientation of the alpha-helices from the linear dichroism of the infrared amide bands, particularly in membranes. The order parameters of the amide vibrational transition moments are calculated for the transmembrane alpha-helices of bacteriorhodopsin by using the crystal structure determined at a resolution of 1.55 A (PDB accession number 1C3W). The dependence on the angle delta(M) that the transition moment makes with the peptide carbonyl bond is fit by the expression ((3)/(2)S(alpha) cos(2) alpha)cos(2)(delta(M) + beta) - 1/2S(alpha), where S(alpha) (0.91) is the order parameter of the alpha-helices, alpha (13 degrees ) is the angle that the peptide plane makes with the helix axis, and beta (11 degrees ) is the angle that the peptide carbonyl bond makes with the projection of the helix axis on the peptide plane. This result is fully consistent with the model of nested axial distributions commonly used in interpreting infrared linear dichroism of proteins. Comparison with experimental infrared dichroic ratios for bacteriorhodopsin yields values of Theta(A) = 33 +/- 1 degree, Theta(I) = 39.5 +/- 1 degree, and Theta(II) = 70 +/- 2 degrees for the orientation of the transition moments of the amide A, amide I, and amide II bands, respectively, relative to the helix axis. These estimates are close to those found for model alpha-helical polypeptides, indicating that side-chain heterogeneity and slight helix imperfections are unlikely to affect the reliability of infrared measurements of helix orientations.     

What spectroscopy can still tell us about the secondary structure of bacteriorhodopsin.

The recently published model of the structure of bacteriorhodopsin (bR), developed by fitting the peptide chain to a high-resolution, three-dimensional density map, rules out the existence of transmembrane beta-sheet and provides an accurate estimate of the helix content. The precise geometry of the dihedral angles in the helical regions of the polypeptide cannot yet be specified from the diffraction data, however. Published data on the circular dichroism (CD) spectrum between 190 and 240 nm, and the infrared (IR) spectrum in the amide I band suggest that the helical conformation in bR may be, for the most part, a rather unusual one. The precise structural model, which specifies the number of residues in transmembrane helices, can now be used as an additional constraint in seeking models of the helical conformation that are in quantitative agreement with the CD and IR spectroscopic data. Further spectroscopic measurements can also be used to determine whether there are changes in the unusual dihedral-angle conformation within the helices during the photocycle.     

Structural change of bacteriorhodopsin in the purple membrane above pH 10 decreases heterogeneity of the irreversible photobleaching components

Kinetic investigations of irreversible photobleaching of bacteriorhodopsin (bR) in purple membrane (PM) at high temperature have previously shown two kinds of bR species upon light illumination. The bR species consist of kinetically fast- and slow-denatured components, whose proportions were dependent upon structural changes in dark, as shown by CD. In order to elucidate electrostatic contribution on the heterogeneous stability and the bR structure in PM, photobleaching behaviour and structural changes over a wide pH range were investigated by kinetics as well as various spectroscopic techniques. Kinetics revealed that photobleaching below pH 9 obeyed double-exponential functions, whereas measurements above pH 10 were characterized by a single-decay component. FT-IR deconvoluted spectra showed a alpha(II)-to-alpha(I) transition in the transmembrane helices around pH 10. Near-IR Raman scattering spectra demonstrated the equilibrium shift of retinal isomers from all trans to 13-cis form. Near-UV CD spectra suggested configurational changes in the aromatic residues around the retinal pocket. An exciton-to-positive transition in visible CD spectrum was observed. This indicates disorganization in the 2D-crystalline lattice of PM, which occurred concomitantly with the changes above pH 10. A model for the changes in kinetic behaviour and molecular structure around pH 10 is discussed, focusing on changes in charge distribution upon alkalinization.     

Tilt angle of a trans-membrane helix is determined by hydrophobic mismatch

In order to investigate the compensation mechanism of a trans-membrane helix in response to hydrophobic mismatch, the tilt and rotation angles of the trans-membrane helix of Vpu aligned in lipid bilayers of various thickness were determined using orientation-dependent frequencies obtained from solid-state NMR experiments of aligned samples. A tilt angle of 18 degrees was observed in 18:1-O-PC/DOPG (9:1) lipid bilayers, which have a hydrophobic thickness that approximately matches the hydrophobic length of the trans-membrane helix of Vpu. Upon decreasing the hydrophobic thickness of lipid bilayers, no significant change in rotation angle was observed. However, the tilt angle increased systematically with increasing positive mismatch to 27 degrees in 14:0-O-PC/DMPG (9:1), 35 degrees in 12:0-O-PC/DLPG (9:1), and 51 degrees in 10:0 PC/10:0 PG (9:1) lipid bilayers, indicating that the change in tilt angle of the trans-membrane helix is a principal compensation mechanism for hydrophobic mismatch. In addition, the distinctive kink in the middle of the helix observed in 18:1 bilayers disappears in thinner bilayers. Although the opposite of what might be expected, this finding suggests that a helix kink may also be a part of the hydrophobic matching mechanism for trans-membrane helices.     

Structure and orientation of lung surfactant SP-C and L-alpha-dipalmitoylphosphatidylcholine in aqueous monolayers.

SP-C, a pulmonary surfactant-specific protein, aids the spreading of the main surfactant phospholipid L-alpha-dipalmitoylphosphatidylcholine (DPPC) across air/water interfaces, a process that has possible implications for in vivo function. To understand the molecular mechanism of this process, we have used external infrared reflection-absorption spectroscopy (IRRAS) to determine DPPC acyl chain conformation and orientation as well as SP-C secondary structure and helix tilt angle in mixed DPPC/SP-C monolayers in situ at the air/water interface. The SP-C helix tilt angle changed from approximately 24 degrees to the interface normal in lipid bilayers to approximately 70 degrees in the mixed monolayer films, whereas the acyl chain tilt angle of DPPC decreased from approximately 26 degrees in pure lipid monolayers (comparable to bilayers) to approximately 10 degrees in the mixed monolayer films. The protein acts as a "hydrophobic lever" by maximizing its interactions with the lipid acyl chains while simultaneously permitting the lipids to remain conformationally ordered. In addition to providing a reasonable molecular mechanism for protein-aided spreading of ordered lipids, these measurements constitute the first quantitative determination of SP-C orientation in Langmuir films, a paradigm widely used to simulate processes at the air/alveolar interface.     

Structure and conformation of the disulfide bond in dimeric lung surfactant peptides SP-B1-25 and SP-B8-25

Raman spectroscopy was used to determine the conformation of the disulfide linkage between cysteine residues in the homodimeric construct of the N-terminal alpha helical domain of surfactant protein B (dSP-B(1-25)). The conformation of the disulfide bond between cysteine residues in position 8 of the homodimer of dSP-B(1-25) was compared with that of a truncated homodimer (dSP-B(8-25)) of the peptide having a disulfide linkage at the same position in the alpha helix. Temperature-dependent Raman spectra of the S-S stretching region centered at approximately 500 cm(-1) indicated a stable, although highly strained disulfide conformation with a chi(CS-SC) dihedral angle of +/-10 degrees for the dSP-B(1-25) dimer. In contrast, the truncated dimer dSP-B(8-25) exhibited a series of disulfide conformations with the chi(CS-SC) dihedral angle taking on values of either +/-30 degrees or 85+/-20 degrees . For conformations with chi(CS-SC) close to the +/-90 degrees value, the Raman spectra of the 8-25 truncated dimers exhibited chi(SS-CC) dihedral angles of 90/180 degrees and 20-30 degrees . In the presence of a lipid mixture, both constructs showed a nu(S-S) band at approximately 488 cm(-1), corresponding to a chi(CS-SC) dihedral angle of +/-10 degrees . Polarized infrared spectroscopy was also used to determine the orientation of the helix and beta-sheet portion of both synthetic peptides. These calculations indicated that the helix was oriented primarily in the plane of the surface, at an angle of approximately 60-70 degrees to the surface normal, while the beta structure had approximately 40 degrees tilt. This orientation direction did not change in the presence of a lipid mixture or with temperature. These observations suggest that: (i) the conformational flexibility of the disulfide linkage is dependent on the amino acid residues that flank the cysteine disulfide bond, and (ii) in both constructs, the presence of a lipid matrix locks the disulfide bond into a preferred conformation.     

Circular dichroism of heterochromophoric and partially regenerated purple membrane: search for exciton coupling

In order to determine the origin of the bisignate CD spectra of native purple membrane, heterochromophoric analogues containing bacteriorhodopsin regenerated with native all-trans-retinal and retinal analogues were investigated. The data collected for the purple membrane samples containing two different chromophores suggest the additive character of the CD spectra. This conclusion was supported by a series of spectra using 5,6-dihydroretinal and 3-dehydroretinal and by using 33% regenerated PM in buffer and in presence of osmolytes. Our results support the idea of conformational heterogeneity of the chromophores in the bR in the trimer, suggesting that the three bR subunits in the trimer are not conformationally equal, and therefore, the bisignate CD spectrum of bR in the purple membrane occurs rather due to a superposition of the CD spectra from variously distorted bR subunits in the trimer than interchromophoric exciton-coupling interactions.     

Effect of acid pH on the absorption spectra and photoreactions of bacteriorhodopsin.

Purple membranes (PM) from Halobacterium halobium were incorporated into 7.5% polyacrylamide gels to prevent aggregation which occurs in suspensions at low pH. At pH 7.0, the circular dichroism (CD) spectra and visible absorption spectra of light- and dark-adapted bacteriorhodopsin (bR558, respectively) and the flash photolysis cycle of bR568 in gels were essentially the same as those in PM suspensions. Titration of the gels with hydrochloric acid showed the transition to a form absorbing at 605 nm (bR605 acid) with pK = 2.9 and to a second form absorbing at 565 nm (bR565 acid) with pK = 0.5. Isosbestic points were seen for each transition in both light- and dark-adapted gels. In addition, a third isosbestic point was evident between pH 3.5 and 7. Visible CD spectra of bR568, bR605 acid, and bR565 acid all showed the bilobed pattern typical of bR568 in suspensions of PM. Flash kinetic spectrophotometry (with 40-microseconds time resolution) of bR605 acid and bR565 acid showed transient absorbance changes with at least one transiently blue-shifted form for each and an early red-shifted intermediate for bR565 acid. Chromophore extraction from membrane suspensions yielded all-trans-retinal for bR565 acid and a mixture of 13-cis and trans isomers for bR605 acid.     

Investigation of the orientation of purple membrane sheets in Langmuir-Blodgett films by a quartz crystal microbalance

By means of the quartz crystal microbalance (QCM), a convenient method was developed to determine the degree of orientation of purple membrane (PM) sheets on the air/water interface. Langmuir-Blodgett films from both wild-type and SH-mutant PM (bR D36C) were vertically deposited on the surface of gold-sputtered AT-cut quartz crystals. The shift of resonance frequency of the QCM during a special washing protocol allowed us to differentiate between physically adsorbed PM fragments and any PM attached to the gold surface via chemical bonds. By washing with organic solvents, complete desorption of the wild-type PM was achieved, whereas for the SH-mutant, approximately 60% of the PM fragments could not be detached from the surface. These PM sheets should be oriented with the cytoplasmic side facing the water subphase to that their SH-groups can chemically bind to the gold surface of the quartz plate.     

vpu transmembrane peptide structure obtained by site-specific fourier transform infrared dichroism and global molecular dynamics searching.

The recently developed method of site-directed Fourier transform infrared dichroism for obtaining orientational constraints of oriented polymers is applied here to the transmembrane domain of the vpu protein from the human immunodeficiency virus type 1 (HIV-1). The infrared spectra of the 31-residue-long vpu peptide reconstituted in lipid vesicles reveal a predominantly alpha-helical structure. The infrared dichroism data of the (13)C-labeled peptide yielded a helix tilt beta = (6.5 +/- 1.7) degrees from the membrane normal. The rotational pitch angle omega, defined as zero for a residue located in the direction of the helix tilt, is omega = (283 +/- 11) degrees for the (13)C labels Val(13)/Val(20) and omega = (23 +/- 11) degrees for the (13)C labels Ala(14)/Val(21). A global molecular dynamics search protocol restraining the helix tilt to the experimental value was performed for oligomers of four, five, and six subunits. From 288 structures for each oligomer, a left-handed pentameric coiled coil was obtained, which best fits the experimental data. The structure reveals a pore occluded by Trp residues at the intracellular end of the transmembrane domain.     

Structure, topology, and tilt of cell-signaling peptides containing nuclear localization sequences in membrane bilayers determined by solid-state NMR and molecular dynamics simulation studies

Cell-signaling peptides have been extensively used to transport functional molecules across the plasma membrane into living cells. These peptides consist of a hydrophobic sequence and a cationic nuclear localization sequence (NLS). It has been assumed that the hydrophobic region penetrates the hydrophobic lipid bilayer and delivers the NLS inside the cell. To better understand the transport mechanism of these peptides, in this study, we investigated the structure, orientation, tilt of the peptide relative to the bilayer normal, and the membrane interaction of two cell-signaling peptides, SA and SKP. Results from CD and solid-state NMR experiments combined with molecular dynamics simulations suggest that the hydrophobic region is helical and has a transmembrane orientation with the helical axis tilted away from the bilayer normal. The influence of the hydrophobic mismatch, between the hydrophobic length of the peptide and the hydrophobic thickness of the bilayer, on the tilt angle of the peptides was investigated using thicker POPC and thinner DMPC bilayers. NMR experiments showed that the hydrophobic domain of each peptide has a tilt angle of 15 +/- 3 degrees in POPC, whereas in DMPC, 25 +/- 3 degree and 30 +/- 3 degree tilts were observed for SA and SKP peptides, respectively. These results are in good agreement with molecular dynamics simulations, which predict a tilt angle of 13.3 degrees (SA in POPC), 16.4 degrees (SKP in POPC), 22.3 degrees (SA in DMPC), and 31.7 degrees (SKP in DMPC). These results and simulations on the hydrophobic fragment of SA or SKP suggest that the tilt of helices increases with a decrease in bilayer thickness without changing the phase, order, and structure of the lipid bilayers.     

Alpha-helix to random coil transitions of two-chain coiled coils: experiments on the thermal denaturation of isolated segments of alpha alpha-tropomyosin

Circular dichroism (CD) experiments in the backbone (200-240 nm) region are reported for four isolated, excised two-chain, coiled-coil segments whose chains comprise, respectively, residues 11-127, 142-281, 1-189, and 190-284 of the rabbit alpha alpha-tropomyosin (Tm) sequence. The uv and CD spectra for the noncross-linked segments are very similar to those for parent Tm. At 3 degrees C, all have a helix content of 90% or more; moreover, all thermal denaturation curves depend on concentration, as required by mass action, and are completely reversible. At comparable concentrations, solutions show values of T1/2 (the temperature at which the helix content is 50%) following the order of 11Tm127 approximately 1Tm189 greater than 142Tm281 greater than 190Tm284. The thermal unfolding data for 11Tm127, 190Tm284, and 142Tm281 fall on apparently monophasic curves (single inflection point). However, curves for 1Tm189 show a heretofore unknown low temperature transition in which the helix content drops from approximately 90% at 2 degrees C to approximately 73% at 20 degrees C, indicating that this segment has one or more weak sections totaling approximately 50 residues per chain. Since thermal denaturation curves for noncross-linked 11Tm127, 142Tm281, and Tm have no such low temperature transition, i.e., the helix content is not additive, the weak region probably comprises the bulk of the residues between 127 and 189 in 1Tm189, but is somehow stabilized in 142Tm281 and in parent Tm.(ABSTRACT TRUNCATED AT 250 WORDS)