Calmodulin and troponin C structures studied by Fourier transform infrared spectroscopy: effects of Ca2+ and Mg2+ binding

Fourier transform infrared (FTIR) spectroscopy has been used to examine the conformationally sensitive amide I' bands of calmodulin and troponin C. These are observed to undergo a sequence of spectroscopic changes which reflect conformational rearrangements that take place when Ca2+ is bound. Calmodulin and troponin C show similar though not identical changes on Ca2+ binding, and the effect of Mg2+ on troponin C is quite different from that of Ca2+. Both proteins show absorption maxima in the amide I' region at 1644 cm-1 which is significantly lower in frequency than has been generally observed for proteins that contain a high percentage of alpha-helix. It is proposed that an unusually high proportion of the helices in the structures of these proteins are distorted from the normal alpha-helical configuration such that the carbonyl stretching frequencies are lowered. It is further proposed that the shift to lower frequency is due to backbone carbonyl groups in the distorted helices that form strong hydrogen bonds with solvent molecules. A decrease in intensity at 1654 cm-1, the normal frequency assignment for alpha-helical structure, is observed as Ca2+ binds to calmodulin and troponin C. This suggests that Ca2+ binding results in a net decrease in "normal" alpha-helix conformation. There is a corresponding increase in intensity of the band at 1644 cm-1, possibly due to an increase in distorted helix content, allowing for a net increase in helix consistent with circular dichroism estimates of the Ca2+-dependent changes in helix content in calmodulin.     

Protein conformational transitions in sarcoplasmic reticulum membranes

Fourier-transform infrared spectroscopic studies of sarcoplasmic reticulum proteins, in H2O and D2O, suggest that 10 mM ATP induces a conformational change in those proteins, increasing their contents in alpha-helical and beta-antiparallel structures. Ca2+ on the contrary, is seen to reduce the proportion of alpha-helix and increase the contribution of random coil.     

Estimation of the number of alpha-helical and beta-strand segments in proteins using circular dichroism spectroscopy

A simple approach to estimate the number of alpha-helical and beta-strand segments from protein circular dichroism spectra is described. The alpha-helix and beta-sheet conformations in globular protein structures, assigned by DSSP and STRIDE algorithms, were divided into regular and distorted fractions by considering a certain number of terminal residues in a given alpha-helix or beta-strand segment to be distorted. The resulting secondary structure fractions for 29 reference proteins were used in the analyses of circular dichroism spectra by the SELCON method. From the performance indices of the analyses, we determined that, on an average, four residues per alpha-helix and two residues per beta-strand may be considered distorted in proteins. The number of alpha-helical and beta-strand segments and their average length in a given protein were estimated from the fraction of distorted alpha-helix and beta-strand conformations determined from the analysis of circular dichroism spectra. The statistical test for the reference protein set shows the high reliability of such a classification of protein secondary structure. The method was used to analyze the circular dichroism spectra of four additional proteins and the predicted structural characteristics agree with the crystal structure data.     

Halogenated alcohols as solvents for proteins: FTIR spectroscopic studies.

Fourier transform infrared (FTIR) spectroscopy has been applied to investigate the secondary structure of proteins and polypeptides in halogenated alcohols. Each alcohol studied was able, as a pure liquid, to induce conversion of the beta-sheet protein concanavalin A into a predominantly alpha-helical configuration. In 2H2O/alcohol mixtures, helicogenisis was also apparent, decreasing in the order dichloroethanol greater than bromoethanol greater than trifluoroethanol greater than chloroethanol greater than fluoroethanol. At concentrations below those found to be helicogenic, disruption of the protein secondary structure by the alcohols resulted in pronounced aggregation. At concentrations insufficient to cause noticeable disruptions of the secondary structure at room temperature, the thermal stability of the protein was greatly reduced. We suggest the helicogenic effect exhibited by halogenated alcohols to be related to a combination of a relatively low dielectric constant and a high dipole moment, the latter causing disruption of the internal hydrogen bond networks and the former causing refolding to a helical configuration. The results presented here highlight the risk of using halogenated alcohols, both as solvents for proteins and as a test of the intrinsic capacity of proteins and peptides to adopt helical secondary structures.     

Peptides derived from apoptotic Bax and Bid reproduce the poration activity of the parent full-length proteins

Bax and Bid are proapoptotic proteins of the Bcl-2 family that regulate the release of apoptogenic factors from mitochondria. Although they localize constitutively in the cytoplasm, their apoptotic function is exerted at the mitochondrial outer membrane, and is related to their ability to form transbilayer pores. Here we report the poration activity of fragments from these two proteins, containing the first alpha-helix of a colicinlike hydrophobic hairpin (alpha-helix 5 of Bax and alpha-helix 6 of Bid). Both peptides readily bind to synthetic lipid vesicles, where they adopt predominantly alpha-helical structures and induce the release of entrapped calcein. In planar lipid membranes they form ion conducting channels, which in the case of the Bax-derived peptide are characterized by a two-stage pattern, a large conductivity and lipid-charge-dependent ionic selectivity. These features, together with the influence of intrinsic lipid curvature on the poration activity and the existence of two helical stretches of different orientations for the membrane-bound peptide, suggest that it forms mixed lipidic/peptidic pores of toroidal structure. In contrast, the assayed Bid fragment shows a markedly different behavior, characterized by the formation of discrete, steplike channels in planar lipid bilayers, as expected for a peptidic pore lined by a bundle of helices.     

Exploration of structural features of monomeric helical peptides designed with a genetic algorithm

A genetic algorithm (GA)-based strategy to dissect the determinants of peptide folding into alpha-helix was developed. The structural information of helical peptides was obtained with respect to patterns of sequence variability. In many previously reported studies the intrinsic alpha-helical propensities of amino acids although sequence-dependent are apparently independent of the amino acid position. In this research, monomeric helical peptides selected from possible sequences produced by a GA-chemical synthesis were analyzed to identify possible influential structural features. These hexadeca-peptides were obtained after four successive generations. A total of 128 synthetic peptides were evaluated via circular dichroism (CD) measurements in aqueous solution, while the mean ellipticity at 222 nm confirmed the monomeric state of the peptides. The results presented here show that our GA-based strategy may be useful in the design of proteins with increased alpha-helix content.     

The secondary structure of apolipoproteins in human HDL3 particles after chemical modification of their tyrosine, lysine, cysteine or arginine residues. A Fourier transform infrared spectroscopy study

Fourier transform infrared spectra of apolipoprotein E-depleted human HDL3 have been obtained in H2O and 2H2O buffers. The absorption bands in the protein amide I and amide II regions (1700-1500 cm-1) were assigned to alpha-helical, disordered and beta-strand/beta-turn structures of apolipoproteins A-I and A-II (apoA-I and apoA-II), the apolipoprotein constituents of HDL3. Modification of HDL3 by tetranitromethane (TNM) treatment, acetylation, reduction plus alkylation and 1,2-cyclohexanedione treatment derivatised tyrosine, lysine, cysteine and arginine residues, respectively, and caused alteration of the secondary structure of the HDL3 apolipoproteins to different extents. Each of the chemical modifications caused changes in the frequency of bands associated with beta-strands/beta-turns, but only TNM treatment of HDL3, as judged by the second- and fourth-derivative spectra, resulted in a shift of the band assigned to the alpha-helical structure of the proteins. In agreement with other workers, only TNM treatment of HDL3 particles was found to inhibit their binding by high-affinity cell membrane receptors. It is proposed, therefore, that receptor recognition of HDL3 particles is dependent on conservation of the alpha-helix structures within apoA-I and apoA-II, and that beta-strand/beta-turn structures are not involved. This conclusion is consistent with the predominance of amphipathic alpha-helical structures in both apolipoproteins and with the relaxed specificity of the receptors which are thought to recognise both apoA-I and apoA-II.     

Positional preference of proline in alpha-helices.

Conformational free energy calculations have been carried out for proline-containing alanine-based pentadecapeptides with the sequence Ac-(Ala)n-Pro-(Ala)m-NHMe, where n + m = 14, to figure out the positional preference of proline in alpha-helices. The relative free energy of each peptide was calculated by subtracting the free energy of the extended conformation from that of the alpha-helical one, which is used here as a measure of preference. The highest propensity is found for the peptide with proline at the N-terminus (i.e., Ncap + 1 position), and the next propensities are found at Ncap, N' (Ncap - 1), and C' (Ccap + 1) positions. These computed results are reasonably consistent with the positional propensities estimated from X-ray structures of proteins. The breaking in hydrogen bonds around proline is found to play a role in destabilizing alpha-helical conformations, which, however, provides the favored hydration of the corresponding N-H and C=O groups. The highest preference of proline at the beginning of alpha-helix appears to be due to the favored electrostatic and nonbonded energies between two residues preceding proline and the intrinsic stability of alpha-helical conformation of the proline residue itself as well as no disturbance in hydrogen bonds of alpha-helix by proline. The average free energy change for the substitution of Ala by Pro in a alpha-helix is computed to be 4.6 kcal/mol, which is in good agreement with the experimental value of approximately 4 kcal/mol estimated for an oligopeptide dimer and proteins of barnase and T4 lysozyme.     

A Fourier transform infrared spectroscopic (FTIR) study of porcine and bovine pancreatic phospholipase A2 and their interaction with substrate analogues and a transition-state inhibitor

Fourier transform infrared spectroscopy has been used to investigate the secondary structure of porcine and bovine pancreatic phospholipase A2 (PLA2) and the zymogen of porcine PLA2, prophospholipase A2 (proPLA2), in both H2O and D2O media. Detailed qualitative analysis was made of these proteins using second derivative and deconvolution techniques. Quantitative studies of the proteins in solution made using Factor Analysis gave average values of 54% alpha-helix, 15% beta-sheet and 23% beta-turns. These values agree well with the secondary structures deduced from previous studies of single crystals using X-ray techniques. No significant differences in secondary structure were observed for porcine pancreatic (pro)phospholipase A2 in the presence or absence of Ca2+ ions, or in the temperature range 10-45 degrees C. The binding of the non-degradable phospholipid analogue, n-alkylphosphocholine, in monomeric form produced no significant difference in the secondary structure of either enzyme. Conformational differences were, however, observed between the enzyme lyophilised in a solid film and in aqueous solution. The change is probably due to the formation of beta-sheet upon hydration, coupled with a loss of random structures. Conformational differences in both porcine and bovine pancreatic PLA2 were observed on binding to n-alkylphosphocholine micelles. This change may be due to a small increase in the alpha-helical structure and a decrease in the beta-sheet, and/or possibly beta-turn content. Similar conformational changes were observed for the interaction of porcine and bovine PLA2 with the substrate analogue inhibitor 1-heptanoyl-2-heptanoylamino-2-deoxy-sn-glycero-3-phospho glycol in micellar form.     

Steric restrictions in protein folding: an alpha-helix cannot be followed by a contiguous beta-strand

Using only hard-sphere repulsion, we investigated short polyalanyl chains for the presence of sterically imposed conformational constraints beyond the dipeptide level. We found that a central residue in a helical peptide cannot adopt dihedral angles from strand regions without encountering a steric collision. Consequently, an alpha-helical segment followed by a beta-strand segment must be connected by an intervening linker. This restriction was validated both by simulations and by seeking violations within proteins of known structure. In fact, no violations were found within an extensive database of high-resolution X-ray structures. Nature's exclusion of alpha-beta hybrid segments, fashioned from an alpha-helix adjoined to a beta-strand, is built into proteins at the covalent level. This straightforward conformational constraint has far-reaching consequences in organizing unfolded proteins and limiting the number of possible protein domains.     

Prediction of protein secondary structure based on physical theory. Histones

Secondary structures of histones H1, H2A, H2B, H3, H4 and H5 have been calculated by the computer program ALB based on a molecular theory of protein secondary structure. The predicted secondary structures of all histones are predominantly alpha-helical. The calculated secondary structure of linker histones H1 and H5 is close to that previously obtained from two-dimensional NMR data. For each of the core histones (H2A, H2B, H3, H4) one long alpha-helix and several short ones have been predicted. These long helices can be identified with rods in the low-resolution electron density map.     

Folding of apocytochrome c in lipid micelles: formation of alpha-helix precedes membrane insertion.

Apocytochrome c, which in aqueous solution is largely unstructured, acquires a highly alpha-helical structure upon interaction with lipid. The alpha-helix content induced in apocytochrome c depends on the lipid system, and this folding process is driven by both electrostatic and hydrophobic lipid-protein interactions. The folding kinetic mechanism of apocytochrome c induced by zwitterionic micelles of lysophosphatidylcholine (L-PC), predominantly driven by hydrophobic lipid-protein interactions, was investigated by fluorescence stopped-flow measurements of Trp 59 and fluorescein-phosphatidylethanolamine-(FPE) labeled micelles, in combination with stopped-flow far-UV circular dichroism. It was found that formation of the alpha-helical structure of apocytochrome c precedes membrane insertion. The unfolded state in solution (U(W)) binds to the micelle surface in a helical conformation (I(S)) and is followed by insertion into the lipid micelle, i.e., formation of the final helical state H(L). Binding of apocytochrome c to the lipid micelle (U(W) --> I(S)) is concurrent with formation of a large fraction (75-100%, depending on lipid concentration) of the alpha-helical structure of the final lipid-inserted state H(L). The highly helical intermediate I(S) is formed on the time scale of 3-12 ms, depending on lipid concentration, and inserts into the lipid micelle (I(S) --> H(L)) in the time range of approximately 200 ms to >1 s, depending on lipid-to-protein ratio. The final lipid-inserted helical state H(L) in L-PC micelles has an alpha-helix content approximately 65% of that of cytochrome c in solution and has no compact stable tertiary structure as revealed by circular dichroism results.     

Structures of revertant signal sequences of Escherichia coli ribose binding protein.

Recently we reported (Yi et al., 1994) that the alpha-helical content of the signal peptide of Escherichia coli ribose binding protein, when determined by circular dichroism (CD) and two-dimensional NMR in trifluoroethanol/water solvent, is higher than that of its nonfunctional mutant signal peptide. In the present investigation, the structures of the signal peptides of two revertant ribose binding proteins in the same solvent were also determined with CD and two-dimensional 1H NMR spectroscopy. According to the CD results, both of these revertant signal peptides showed an intermediate helicity between those of wild-type and mutant signal peptides, the helical content of the revertant peptide with higher recovery of the translocation capability being higher. On the other hand, the alpha-helix regions of the wild-type and the revertant peptides as determined by NMR were shown to be the same. This discrepancy may be due to the difference in stability between identical alpha-helical stretches in wild-type and revertant peptides. A good correlation was observed between the helical content of these four ribose binding protein signal peptides in TFE/water as studied by CD and their in vivo translocation activities. It appears, therefore, that both the proper length of the helix and the stability are of functional significance.     

Orientation of melittin in phospholipid bilayers. A polarized attenuated total reflection infrared study.

The helical order parameter of the 26-residue amphiphilic bee venom peptide melittin was measured by polarized attenuated total reflection infrared spectroscopy (ATR-IR) in dry phospholipid multibilayers (MBLs) and when bound to single supported planar bilayers (SPBs) under D2O. Melittin adopted an alpha-helical conformation in MBLs of dipalmitoyl-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), a 4:1 mixture of POPC and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), and when bound to SPBs of POPC:POPG (4:1). The order parameter of the alpha-helix in the bilayers depended mainly on the type of membrane preparation, and only little on the phospholipid composition of the bilayers. On hydrated SPBs, the helical order parameter was negative, indicating that the alpha-helix long axis of melittin was preferentially oriented parallel to the plane of the supported membrane. However, in dry MBLs, the helical order parameter was positive, indicating that the alpha-helix of melittin was preferentially oriented parallel to the phospholipid fatty acyl chains. It is concluded that the orientation of melittin in membranes depends on the degree of hydration of the model membranes rather than on the technique which is used for its determination. ATR-IR spectroscopy of polypeptides in or associated with supported planar membranes in D2O may become a useful tool for the determination of their orientation in and on membranes.     

Secondary structural analysis of two recombinant murine proteins, interleukins 1 alpha and 1 beta: is infrared spectroscopy sufficient to assign structure?

The secondary structure for two murine recombinant proteins, interleukins 1 alpha and 1 beta (rmIL-1 alpha and -1 beta), has been analyzed by Fourier transform infrared (IR) spectroscopy and then compared to results obtained by X-ray diffraction, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy. The IR results obtained here for rmIL-1 alpha and -1 beta suggested that their secondary structures consisted predominantly of beta-sheets or strands. However, the analysis also revealed a significant absorption band near 1656 cm-1, which is typically assigned to alpha-helical or random structures. When these same murine polypeptides were analyzed by CD, no evidence of alpha-helical structures was observed. Further, published X-ray diffraction and NMR studies characterizing the human forms of IL-1 alpha and -1 beta indicate the absence of alpha-helices and that the human proteins are composed mainly of beta-strands (i.e., greater than 55%), with approximately 24% of the amino acids involved in large loops connecting the strands. The murine IL-1 proteins, when compared to their respective human counterparts, each show greater than 80% sequence homology. Given this fact, the CD analyses, and the result that this IR band amounted to 21% of the overall integrated area, the absorption peak at 1656 cm-1 was attributed to the presence of large loops rather than to alpha-helical or random structures. Such a structural assignment appears reasonable and is totally consistent with the established existence of large loops in the human forms as well as in other proteins found to fold similarly (viz., human bFGF).(ABSTRACT TRUNCATED AT 250 WORDS)     

Addition of side-chain interactions to 3(10)-helix/coil and alpha-helix/3(10)-helix/coil theory.

An increasing number of experimental and theoretical studies have demonstrated the importance of the 3(10)-helix/ alpha-helix/coil equilibrium for the structure and folding of peptides and proteins. One way to perturb this equilibrium is to introduce side-chain interactions that stabilize or destabilize one helix. For example, an attractive i, i + 4 interaction, present only in the alpha-helix, will favor the alpha-helix over 3(10), while an i, i + 4 repulsion will favor the 3(10)-helix over alpha. To quantify the 3(10)/alpha/coil equilibrium, it is essential to use a helix/coil theory that considers the stability of every possible conformation of a peptide. We have previously developed models for the 3(10)-helix/coil and 3(10)-helix/alpha-helix/ coil equilibria. Here we extend this work by adding i, i + 3 and i, i + 4 side-chain interaction energies to the models. The theory is based on classifying residues into alpha-helical, 3(10)-helical, or nonhelical (coil) conformations. Statistical weights are assigned to residues in a helical conformation with an associated helical hydrogen bond, a helical conformation with no hydrogen bond, an N-cap position, a C-cap position, or the reference coil conformation plus i, i + 3 and i, i + 4 side-chain interactions. This work may provide a framework for quantitatively rationalizing experimental work on isolated 3(10)-helices and mixed 3(10)-/alpha-helices and for predicting the locations and stabilities of these structures in peptides and proteins. We conclude that strong i, i + 4 side-chain interactions favor alpha-helix formation, while the 3(10)-helix population is maximized when weaker i, i + 4 side-chain interactions are present.     

Protein structures in SDS micelle-protein complexes.

Sodium dodecyl sulfate (SDS) is used more often than any other detergent as an excellent denaturing or "unfolding" detergent. However, formation of ordered structure (alpha-helix or beta-sheet) in certain peptides is known to be induced by interaction with SDS micelles. The SDS-induced structures formed by these peptides are amphiphilic, having both a hydrophobic and a hydrophilic face. Previous work in this area has revealed that SDS induces helical folding in a wide variety of non-helical proteins. Here, we describe the interaction of several structurally unrelated proteins with SDS micelles and the correlation of these structures to helical amphiphilic regions present in the primary sequence. It is likely that the ability of native nonordered protein structures to form induced amphiphilic ordered structures is rather common.     

Indolicidin,a 13-residue basic antimicrobial peptide rich in tryptophan and proline,interacts with Ca(2+)-calmodulin

Indolicidin, ILPWKWPWWPWRR-NH(2), a short 13-residue antimicrobial and cytolytic peptide characterized from bovine neutrophils, has the calmodulin-recognition 1-5-10 hydrophobic pattern (indicated by amino acids in bold), is cationic, and thereby fulfills the requirements to interact with calmodulin. Hence, we have investigated the calmodulin-binding properties of indolicidin. Indolicidin interacted with calmodulin with fairly high affinity in a Ca(2+)-dependent manner. However, when bound, the peptide did not adopt helical conformation. Indolicidin also inhibited calmodulin-stimulated phosphodiesterase activity with IC(50) values in the nanomolar range. Replacement of either the proline residues of indolicidin with alanines or tryptophan residues with phenylalanines did not affect binding to calmodulin. However, these replacements had distinctive effects on the conformations of the bound peptides. While the alanine analog of indolicidin adopted predominantly alpha-helical conformation, the phenylalanine analog remained largely unordered. Differences in the ability of these analogs to inhibit the calmodulin-stimulated phosphodiesterase activity were observed. While the alanine analog was capable of inhibiting the activity with IC(50) values comparable to that of indolicidin, the phenylalanine analog did not inhibit the activity. Our results indicate that ability to adopt amphiphilic alpha-helical structure is not a prerequisite for binding to calmodulin and also binding does not necessarily result in inhibition of calmodulin-stimulated enzyme activities.