Two peptide fragments G55-I72 and K97-A109 from staphylococcal nuclease exhibit different behaviors in conformational preferences for helix formation

Two synthetic peptides, SNasealpha1 and SNasealpha2, corresponding to residues G55-I72 and K97-A109, respectively, of staphylococcal nuclease (SNase), are adopted for detecting the role of helix alpha1 (E57-A69) and helix alpha2 (M98-Q106) in the initiation of folding of SNase. The helix-forming tendencies of the two SNase peptide fragments are investigated using circular dichroism (CD) and two-dimensional (2D) nuclear magnetic resonance (NMR) methods in water and 40% trifluoroethanol (TFE) solutions. The coil-helix conformational transitions of the two peptides in the TFE-H2O mixture are different from each other. SNasealpha1 adopts a low population of localized helical conformation in water, and shows a gradual transition to helical conformation with increasing concentrations of TFE. SNasealpha2 is essentially unstructured in water, but undergoes a cooperative transition to a predominantly helical conformation at high TFE concentrations. Using the NMR data obtained in the presence of 40% TFE, an ensemble of alpha-helical structures has been calculated for both peptides in the absence of tertiary interactions. Analysis of all the experimental data available indicates that formation of ordered alpha-helical structures in the segments E57-A69 and M98-Q106 of SNase may require nonlocal interactions through transient contact with hydrophobic residues in other parts of the protein to stabilize the helical conformations in the folding. The folding of helix alpha1 is supposed to be effective in initiating protein folding. The formation of helix alpha2 depends strongly on the hydrophobic environment created in the protein folding, and is more important in the stabilization of the tertiary conformation of SNase.     

Folding propensities of peptide fragments of myoglobin.

Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthetized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformation preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the alpha region of (phi, psi) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A- and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate beta-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, either studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding.     

Folding propensities of synthetic peptide fragments covering the entire sequence of phage 434 Cro protein.

The phage 434 Cro protein, the N-terminal domain of its repressor (R1-69) and that of phage lambda (lambda6-85) constitute a group of small, monomeric, single-domain folding units consisting of five helices with striking structural similarity. The intrinsic helix stabilities in lambda6-85 have been correlated to its rapid folding behavior, and a residual hydrophobic cluster found in R1-69 in 7 M urea has been proposed as a folding initiation site. To understand the early events in the folding of 434 Cro, and for comparison with R1-69 and lambda6-85, we examined the conformational behavior of five peptides covering the entire 434 Cro sequence in water, 40% (by volume) TFE/water, and 7 M urea solutions using CD and NMR. Each peptide corresponds to a helix and adjacent residues as identified in the native 434 Cro NMR and crystal structures. All are soluble and monomeric in the solution conditions examined except for the peptide corresponding to the 434 Cro helix 4, which has low water solubility. Helix formation is observed for the 434 Cro helix 1 and helix 2 peptides in water, for all the peptides in 40% TFE and for none in 7 M urea. NMR data indicate that the helix limits in the peptides are similar to those in the native protein helices. The number of side-chain NOEs in water and TFE correlates with the helix content, and essentially none are observed in 7 M urea for any peptide, except that for helix 5, where a hydrophobic cluster may be present. The low intrinsic folding propensities of the five helices could account for the observed stability and folding behavior of 434 Cro and is, at least qualitatively, in accord with the results of the recently described diffusion-collision model incorporating intrinsic helix propensities.     

Role of the helical structure of the N-terminal region of Plasmodium falciparum merozoite surface protein 2 in fibril formation and membrane interaction

Merozoite surface protein 2 (MSP2), an abundant glycosylphosphatidylinositol-anchored protein on the surface of Plasmodium falciparum merozoites, is a promising malaria vaccine candidate. MSP2 is intrinsically disordered and forms amyloid-like fibrils in solution under physiological conditions. The 25 N-terminal residues (MSP2(1-25)) play an important role in both fibril formation and membrane binding of the full-length protein. In this study, the fibril formation and solution structure of MSP2(1-25) in the membrane mimetic solvents sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), and trifluoroethanol (TFE) have been investigated by transmission electronic microscopy, turbidity, thioflavin T fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy. Turbidity data showed that the aggregation of MSP2(1-25) was suppressed in the presence of membrane mimetic solvents. CD spectra indicated that helical structure in MSP2(1-25) was stabilized in SDS and DPC micelles and in high concentrations of TFE. The structure of MSP2(1-25) in 50% aqueous TFE, determined using NMR, showed that the peptide formed an amphipathic helix encompassing residues 10-24. Low concentrations of TFE favored partially folded helical conformations, as demonstrated by CD and NMR, and promoted MSP2(1-25) fibril formation. Our data suggest that partially folded helical conformations of the N-terminal region of MSP2 are on the pathway to amyloid fibril formation, while higher degrees of helical structure stabilized by high concentrations of TFE or membrane mimetics suppress self-association and thus inhibit fibril formation. The roles of the induced helical conformations in membrane interactions are also discussed.     

Characterization of the structure and dynamics of mastoparan-X during folding in aqueous TFE by CD and NMR spectroscopy

Mastoparan-X, a 14-residue peptide found in wasp venom, does not adopt a well-defined structure in water, but it folds into an alpha-helix upon addition of trifluoroethanol (TFE). At low levels of TFE, the peptide is partially folded, passing through intermediate stages of folding as the amount of TFE is increased. These partially folded states have been characterized by CD and NMR spectroscopy, and methods to estimate the helical content from CD, chemical shift, and nuclear overhauser effect (NOE) data are compared. Variation in the sign and intensity of NOE cross-peaks is observed in different regions of the peptide, indicative of greater mobility of the sidechains compared to the backbone of the peptide. Furthermore, variation in the sidechain mobility is observed, both between sidechains of different amino acids and within the sidechain of a given amino acid. By monitoring chemical shifts and NOE intensities as the TFE concentration is increased, the initiation site for helix formation could be identified. Furthermore, details of the peptide structure and dynamics during the folding process were elucidated.     

Solution structure of the first and second transmembrane segments of the mitochondrial oxoglutarate carrier

The structures of the first and the second transmembrane segment of the bovine mitochondrial oxoglutarate carrier (OGC) were studied by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. Peptides 21–46 and 78–108 of its primary sequence were synthesized and structurally characterized in membrane-mimetic environments. CD data showed that at high concentrations of TFE (>50%) and SDS (>2%) both peptides assume α-helical structures, whereas in more hydrophilic environments only peptide 78–108 has a helical structure. ¹H-NMR spectra of the two peptides in TFE/water and SDS were fully assigned, and the secondary structures of the peptides were obtained from nuclear Overhauser effects, ³J_αH-NH coupling constants and αH chemical shifts. The three-dimensional solution structures of the peptides in TFE/water were generated by distance geometry calculations. A well-defined α-helix was found in the region K24-V39 of peptide 21–46 and in the region A86–F106 of peptide 78–108. We cannot exclude that in intact OGC the extension of these helices is longer. The helix of peptide 21–46 is essentially hydrophobic, whereas that of peptide 78–108 is predominantly hydrophilic.     

Biophysical studies on fragments of the α-factor receptor protein

The receptor for the α-factor mating pheromone of the yeast Saccharomyces cerevisiae consists of 431 amino acid residues and is a member of a family of membrane proteins predicted to have seven transmembrane helices. Fragments of the receptor corresponding to two of the transmembrane helices [residues 246–269 (M6) and 273–302 (M7)], two of the interhelical loops [residues 107–125 (E2) and 191–206 (E3)], and to a portion of the carboxyl terminus [residues 350–372 (CT)] were synthesized using solid-phase methodologies and purified to near homogeneity. CD was used to characterize the secondary structure of these peptides in trifluoroethanol (TFE), in TFE/water mixtures, in sodium dodecyl sulfate (SDS), and in the presence of dimyristoyl phosphatidylcholine (DMPC) liposomes. In TFE, M6 and M7 exhibited CD spectra consistent with highly helical peptides, whereas CT was partially helical. In contrast, E2 and E3 were either disordered or aggregated in this solvent. M6 did not partition well into DMPC vesicles whereas M7 remained helical. Both M6 and M7 assumed helical conformations in 25 mM SDS. The loop neptides and the carboxyl terminus peptide were either in a β-structure or disordered in the presence of lipid. These findings represent the first biophysical evidence for conformations assumed by specific segments of the STE2 receptor protein. © 1994 John Wiley & Sons, Inc.     

Helical structure of dermaseptin B2 in a membrane-mimetic environment

Dermaseptins are antimicrobial peptides from frog skin that have high membrane-lytic activity against a broad spectrum of microorganisms. The structure of dermaseptin B2 in aqueous solution, in TFE/water mixtures, and in micellar and nonmicellar SDS was analyzed by CD, FTIR, fluorescence, and NMR spectroscopy combined with molecular dynamics calculations. Dermaseptin B2 is unstructured in water, but helical conformations, mostly in segment 3-18, are stabilized by addition of TFE. SDS titration showed that dermaseptin B2 assumes nonhelical structures at SDS concentrations far below the critical micellar concentration and helical structures at micellar concentrations. Dermaseptin B2 bound to SDS micelles (0.4 mM peptide, 80 mM SDS) adopts a well-defined amphipathic helix between residues 11-31 connected to a more flexible helical segment spanning residues 1-8 by a flexible hinge region around Val9 and Gly10. Experiments using paramagnetic probes showed that dermaseptin B2 lies near the surface of SDS micelles and that residue Trp3 is buried in the SDS micelle, but close to the surface. A slow exchange equilibrium occurs at higher peptide/SDS ratios (2 mM peptide, 80 mM SDS) between forms having distinct sets of resonances in the N-terminal 1-11 segment. This equilibrium could reflect different oligomeric states of dermaseptin B2 interacting with SDS micelles. Structure-activity studies on dermaseptin B2 analogues showed that the N-terminal 1-11 segment is an absolute requirement for antibacterial activity, while the C-terminal 10-33 region is also important for full antibiotic activity.     

Comparison of the effects of 2,2,2-trifluoroethanol on peptide and protein structure and function

The co-solvent 2,2,2-trifluoroethanol (TFE) has been often used to aid formation of secondary structure in solution peptides or alternately as a denaturant within protein folding studies. Hen egg white lysozyme (HEWL) and a synthetic model peptide defining HEWL helix-4 were used as comparative model systems to systematically investigate the effect of increasing TFE concentrations on the structure of proteins and peptides. HEWL was analyzed using NMR, far-UV CD and fluorescence spectroscopy; with correlation of these results towards changes in enzymatic activity and the helix-4 peptide was analysed using NMR. Data illustrates two conflicting modes of interaction: Low TFE concentrations stabilize tertiary structure, observed from an increase in the number of NMR NOE contacts. Higher TFE concentrations denatured HEWL with the loss of lysozyme tertiary structure. The effects of TFE upon secondary structural elements within HEWL are distinct from those observed for the helix-4 peptide. This illustrates a dissimilar interaction of TFE towards both protein and peptide at equivalent TFE concentrations. The concentration that TFE promotes stabilization over denaturation is likely to be protein dependent although the structural action can be extrapolated to other protein systems with implications for the use of TFE in structural stability studies.     

Hints of nonhierarchical folding of acidic fibroblast growth factor

We have analyzed by circular dichroism (CD) and proton nuclear magnetic resonance (NMR) the helical propensity of the all-beta protein acidic fibroblast growth factor (aFGF) and two peptides corresponding to beta-strand 8 (beta8 peptide, amino acids 95-107) and the beta-strand 8/turn/beta-strand 9 hairpin (beta8/9 peptide, amino acids 95-114), which has been involved in receptor binding. A secondary structure prediction of aFGF carried out by several procedures labels the 95-104 sequence as predominantly alpha-helical. A titration of aFGF with 2,2,2-trifluoroethanol (TFE) induces a change in the far-UV CD spectrum of the protein giving rise to a prominent alpha-helical shape (22% alpha-helix). The cooperativity of the transition and the moderate TFE concentrations used (midpoint at 24%) suggest that the effect of TFE is specific. Moreover, a titration performed at pH 2 yields a higher amount of alpha-helix (55%) at a smaller TFE concentration. Synthetic peptides containing the beta8 and beta8/9 sequences display a random coil conformation at pH 7 but acquire alpha-helical structure in the presence of TFE, methanol, and SDS micelles. At pH below 3.0 a significant amount (20-30%) of alpha-helical conformation is present in both the beta8 and beta8/9 peptides even in the absence of other solvent additives. The secondary structure of the peptides was determined by proton nuclear magnetic resonance (1H NMR). These results suggest that the 95-114 sequence of aFGF has helical propensity and that the protein may fold nonhierarchically in the early steps of folding, acquiring its final beta-structure by a later interaction with the rest of the polypeptide.     

CD and NMR investigations on trifluoroethanol-induced step-wise folding of helical segment from scorpion neurotoxin.

A 14 amino acid residue peptide from the helical region of Scorpion neurotoxin has been structurally characterized using CD and NMR spectroscopy in different solvent conditions. 2,2,2-Trifluoroethanol (TFE) titration has been carried out in 11 steps from 0 to 90% TFE and the gradual stabilization of the conformation to form predominantly alpha-helix covering all of the 14 residues has been studied by 1H and 13C NMR spectroscopy. Detailed information such as coupling constants, chemical shift indices, NOESY peak intensities and amide proton temperature coefficients at each TFE concentration has been extracted and analysed to derive the step-wise preferential stabilization of the helical segments along the length of the peptide. It was found that there is a finite amount of the helical conformation in the middle residues 5-11 even at low TFE concentrations. It was also observed that > 75% TFE (v/v) is required for the propagation of the helix to the N and C termini and for correct packing of the side chains of all of the residues. These observations are significant to understanding the folding of this segment in the protein and may throw light on the inherent preferences and side chain interactions in the formation of the helix in the peptide.     

Solution structure of the first and second transmembrane segments of the mitochondrial oxoglutarate carrier

The structures of the first and the second transmembrane segment of the bovine mitochondrial oxoglutarate carrier (OGC) were studied by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. Peptides 21-46 and 78-108 of its primary sequence were synthesized and structurally characterized in membrane-mimetic environments. CD data showed that at high concentrations of TFE (>50%) and SDS (>2%) both peptides assume alpha-helical structures, whereas in more hydrophilic environments only peptide 78-108 has a helical structure. (1)H-NMR spectra of the two peptides in TFE/water and SDS were fully assigned, and the secondary structures of the peptides were obtained from nuclear Overhauser effects, (3)J(alphaH-NH) coupling constants and alphaH chemical shifts. The three-dimensional solution structures of the peptides in TFE/water were generated by distance geometry calculations. A well-defined alpha-helix was found in the region K24-V39 of peptide 21-46 and in the region A86-F106 of peptide 78-108. We cannot exclude that in intact OGC the extension of these helices is longer. The helix of peptide 21-46 is essentially hydrophobic, whereas that of peptide 78-108 is predominantly hydrophilic.     

3D-Structure of the interior fusion peptide of HGV/GBV-C by 1H NMR, CD and molecular dynamics studies

In this work, we present a structural characterization of the putative fusion peptide E2(279-298) corresponding to the E2 envelope protein of the HGV/GBV-C virus by (1)H NMR, CD and MD studies performed in H(2)O/TFE and in lipid model membranes. The peptide is largely unstructured in water, whereas in H(2)O/TFE and in model membranes it adopts an helical structure (approximately 65-70%). The partitioning free energy DeltaG ranges from -6 to -7.5 kcal mol(-1). OCD measurements on peptide-containing hydrated and oriented lipid multilayers showed that the peptide adopts a predominantly surface orientation. The (1)H NMR data (observed NOEs, deuterium exchange rates, Halpha chemical shift index and vicinal coupling constants) and the molecular dynamics calculations support the conclusions that the peptide adopts a stable helix in the C-terminal 9-18 residues slightly inserted into the lipid bilayer and a major mobility in the amino terminus of the sequence (1-8 residues).     

Circular dichroism studies of helical oligopeptides. Can 3(10) and alpha-helical conformations be chiroptically distinguished?

Circular dichroism studies of seven helical oligopeptides containing alpha-aminoisobutyric acid (Aib) in methanol and trifluoroethanol (TFE) solutions are reported. Peptides ranging from 10 to 21 residues in length have been examined. In all cases distinct negative CD bands characteristic of helical peptides are obtained at approximately 220 and 205 nm corresponding to the n-pi and pi-pi transitions, respectively. The ratio R = [theta] pi-pi is less than 1.0 for all peptides studied. Using crystal structure and n.m.r. results for a 10 residue 3(10) helical peptide and literature values for an alpha-helical 11-residue peptide, it is shown that both helical conformations yield R values of approximately 0.8 in alcoholic solvents. The CD data are considered in the light of 1H n.m.r. studies on these oligopeptides. The results suggest that 3(10) and alpha-helical conformations cannot be distinguished by CD methods.     

Conformational requirement of signal sequences functioning in yeast: circular dichroism and 1H nuclear magnetic resonance studies of synthetic peptides

Recently, we have designed a series of simplified artificial signal sequences and have shown that a proline residue in the signal sequence plays an important role in the secretion of human lysozyme in yeast, presumably by altering the conformation of the signal sequence [Yamamoto, Y., Taniyama, Y., & Kikuchi, M. (1989) Biochemistry 28, 2728-2732]. To elucidate the conformational requirement of the signal sequence in more detail, functional and nonfunctional signal sequences connected to the N-terminal five residues of mature human lysozyme were chemically synthesized and their conformations in a lipophilic environment [aqueous trifluoroethanol (TFE) or sodium dodecyl sulfate micelles] analyzed by circular dichroism (CD) and 1H nuclear magnetic resonance (NMR) spectroscopy. The helix content of the peptides, including functional (L8, CL10) and nonfunctional (L8PL, L8PG, L8PL2) signal sequences, was estimated from CD spectra to be 40-50% and 60-70%, respectively, indicating that the helical structure is more abundant in the nonfunctional signal sequences. Two-dimensional NMR analyses in 50% TFE/H2O revealed that each peptide adopted a helical conformation throughout the sequence except for a few residues at the N- and C-termini. Furthermore, H-D exchange experiments indicated that the helical structure of the C-terminal region of the functional signal sequences (L8 and CL10) was less stable than that of the nonfunctional signal sequences (L8PL and L8PL2). On the basis of these results, a model was developed in which the functional signal sequence is inserted in the membrane with a helical conformation and the C-terminal helix unraveled in an extended conformational form through an interaction with the signal peptidase.     

T178 deletion impairs intermolecular interaction of the peptide Nramp1(164–191)

Natural resistance associated macrophage protein 1 (Nramp1), an integral membrane protein with 12 predicted transmembrane domains (TMs), is a divalent cation transporter associated with infectious and autoimmune diseases. A naturally occurring mutation G169D within TM4 of Nramp1 leads to the loss of function, suggesting potential importance of TM4 for the biological function of the protein. In this study, we determine the three‐dimensional structure and topology of a synthetic peptide, del(T178), corresponding to Nramp1(164‐191) (basically consisting of the putative TM4 of Nramp1) with Thr178 deletion in TFE and SDS micelles using NMR and CD spectroscopic techniques, and compare the results with those of the wildtype peptide. Similarly to the wildtype peptide, the del(T178) peptide still forms an amphiphilic‐like α‐helical structure in both membrane mimics and is embedded in SDS micelles. Differently, whereas the wild‐type peptide forms a helix bundle with the hydrophilic side facing the interior of the bundle, the del(T178) peptide exists as a monomer in the membrane mimics and the hydrophilic side of the helix is located near the interface of SDS micelles. Moreover, a strongly cooperative protonation occurs between intramolecular Asp residues for the del(T178) peptide in SDS micelles, while the cooperative proton binding between intermolecular Asp residues was observed for the wildtype peptide. The difference in the results of the two peptides suggests that the deletion of Thr178 impairs intermolecular interaction of the peptide. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Comparison of the solution conformations of a cell-adhesive peptide LBE and its reverse sequence EBL

T-cell adhesion is mediated by an ICAM-1/LFA-1 interaction; this interaction plays a crucial role in T-cell activation during immune response. LBE peptide, which is derived from the beta-subunit of LFA-1, has been shown to inhibit ICAM-1/LFA-1-mediated T-cell adhesion. In this work, we studied the solution conformations of LBE peptide and its reverse sequence (EBL) by NMR, CD and molecular dynamics simulations. Reverse peptides have been used as controls in biological studies. The effect of reversing the sequence of LBE to EBL peptides on their respective conformations is important in understanding their biological properties in vitro or in vivo. The NMR studies for these peptides were carried out in water and in TFE/water solvent systems. In 40% TFE/water, both peptides exhibited helical conformation. CD studies suggested that the LBE exhibits 30% helical conformation, while the EBL exhibits 20% helical conformation. From the NMR and MD simulation studies, it was evident that the peptides exhibited a stable helical conformation; a stable helical structure was found at Leu6 to Leu15 for LBE and at Gly9 to Leu17 for EBL. The helical conformations of LBE and EBL may be in equilibrium with other possible conformers; the other conformers contain loop and turn structures. Both peptides bind to divalent cations because the LBE is derived from the cation-binding region of the LFA-1. This study shows that reversing the peptide sequence did not alter the secondary structure of the corresponding sequence. Hence, caution must be exercised when using reverse peptides as controls in biological studies. This report will improve our ability to design a better inhibitor of ICAM-1/LFA-1 interaction.     

Peptide models of folding initiation sites of bovine beta-lactoglobulin: identification of nativelike hydrophobic interactions involving G and H strands

In an attempt to characterize the early folding events in bovine beta-lactoglobulin (BLG), a set of peptides, covering the flexible N-terminal region and the stable C-terminus beta-core, was synthesized and analyzed by circular dichroism and by nuclear magnetic resonance in water, trifluoroethanol (TFE), and sodium dodecyl sulfate (SDS) below and above the critical micellar concentration. The role of local and long-range hydrophobic interactions in guiding the folding has been investigated. For the peptide fragment covering the more flexible N-terminal region of BLG (beta-strands A, B), where both theoretical predictions and kinetic refolding experiments suggested the formation of non-native alpha-helix, no native long-range contacts were identified, and a helical secondary structure was stabilized only in the presence of 25 mM SDS. At variance, in 50% (v/v) TFE, native, long-range hydrophobic interactions were observed in the peptide covering the core region comprising G and H beta-strands. The side chains involved in these interactions form a nativelike hydrophobic cluster, thus suggesting that the GH region may act as the folding initiation site for BLG. This result is reinforced by the identification, in the urea denaturated BLG, of residual structure located at the level of the GH interface, as evidenced by NMR analysis. These results, in excellent agreement with kinetic, thermodynamic, and cold denaturation folding data, once more underline the utmost importance of the GH region for the stability and folding of BLG. Severe aggregation effects prevented the structural analysis of the peptide covering the EFGH region, indicating that this larger segment does not represent an independent folding domain and that the terminal alpha-helix is necessary for stabilizing the BLG folding core.     

Solution structure of the fifth and sixth transmembrane segments of the mitochondrial oxoglutarate carrier

The structures of the fifth and sixth transmembrane segments of the bovine mitochondrial oxoglutarate carrier (OGC) and of the hydrophilic loop that connects them were studied by CD and NMR spectroscopies. Peptides F215-R246, W279-K305 and P257-L278 were synthesized and structurally characterized. CD data showed that at high concentrations of TFE and SDS all peptides assume α-helical structures. ¹H-NMR spectra of the three peptides in TFE/water were fully assigned and the secondary structures of the peptides were obtained from nuclear Overhauser effects, ³J_αH-NH coupling constants and αH chemical shifts. The three-dimensional solution structures of the peptides were generated by distance geometry calculations. A well-defined α–helix was found in the region L220-V243 of peptide F215-R246 (TMS-V), in the region P284-M303 of peptide W279-K305 (TMS-VI) and in the region N261-F275 of peptide P257-L278 (hydrophilic loop). The helix L220-V243 exhibited a sharp kink at P239, while a little bend around P291 was observed in the helical region P284-M303. Fluorescence studies performed on peptide W279-K305, alone and together with other transmembrane segments of OGC, showed that the W279 fluorescence was quenched upon addition of peptide F215-R246, but not of peptides K21-K46, R78-R108 and P117-A149 suggesting a specific interaction between TMS-V and TMS-VI of OGC.     

Conformational analysis of a mitochondrial presequence derived from the F1-ATPase beta-subunit by CD and NMR spectroscopy.

Previous studies on mitochondrial targeting presequences have indicated that formation of an amphiphillic helix may be required for efficient targeting of the precursor protein into mitochondria, but the structural details are not well understood. We have used CD and NMR spectroscopy to characterize in detail the structure of a synthetic peptide corresponding to the presequence for the beta-subunit of F1-ATPase, a mitochondrial matrix protein. Although this peptide is essentially unstructured in water, alpha-helix formation is induced when the peptide is placed in structure-promoting environments, such as SDS micelles or aqueous trifluoroethanol (TFE). In 50% TFE (by volume), the peptide is in dynamic equilibrium between random coil and alpha-helical conformations, with a significant population of alpha-helix throughout the entire peptide. The helix is somewhat more stable in the N-terminal part of the presequence (residues 4-10), and this result is consistent with the structure proposed previously for the presequence of another mitochondrial matrix protein, yeast cytochrome oxidase subunit IV. Addition of increasing amounts of TFE causes the alpha-helical content to increase even further, and the TFE titration data for the presequence peptide of the F1-ATPase beta-subunit are not consistent with a single, cooperative transition from random coil to alpha-helix. There is evidence that helix formation is initiated in two different regions of the peptide. This result helps to explain the redundancy of the targeting information contained in the presequence for the F1-ATPase beta-subunit.