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.

Comparison of helix stability in wild-type and mutant LamB signal sequences.

Previous studies of isolated peptides corresponding to the wild-type signal sequence of the LamB protein of Escherichia coli and to several export-impaired mutants demonstrated that a high tendency to adopt an alpha-helical conformation in low dielectric environments was a property of functional sequences. We have now used nuclear magnetic resonance to establish further characteristics of the helical conformation of these signal peptides in a solvent mixture (50% trifluoroethanol, by volume, in water) which mimics the conformational distribution of these peptides in lipid vesicles. The interactions of signal sequences in vivo may depend on the location of the helix in the sequence, on the length of the helical segment, and on the stability of the helix. We find that the hydrophobic core has the most persistent helix conformation and that the stability of this helix correlates with in vivo function of different mutants of the LamB signal sequence. In the family of signal peptides studied here, the length of the helix required for function appears to be less rigidly restricted since a signal peptide from a functional pseudorevertant with 4 residues deleted from the hydrophobic core takes up helix as stably as wild type but incorporates fewer residues in the helix.

Two-dimensional J-resolved proton NMR spectroscopy of oligomannosidic glycopeptides.

Individual anomeric protons that are unresolved in the one-dimensional 250-MHz spectra of oligomannosidic glycopeptides can be separated and characterized by two-dimensional J-resolved NMR spectroscopy. Homogeneous preparations of ovalbumin glycopeptides Man6GlcNAc2Asn and Man5GlcNAc2Asn were characterized by chemical methods, conventional proton NMR, and two-dimensional J-resolved NMR. Due to characteristic differences in coupling constants, mannose (J1,2 less than or equal to 2 Hz) and N-acetylglucosamine (J1,2 approximately 9 Hz) anomeric signals of similar chemical shift were readily separated and identified in the two-dimensional spectra. It is shown that two-dimensional J-resolved NMR spectroscopy, in combination with conventional NMR and limited chemical analysis, is a rapid and reliable technique for the determination of glycopeptide primary structure.

Helix formation and stability in a signal sequence.

A detailed nuclear magnetic resonance analysis of the isolated LamB signal peptide (MMITLRKLPLAVAVAAGVMSAQAMA) under conditions defined by circular dichroism spectra to mimic the conformational distribution of this peptide in membranelike environments has provided a description of specific residue conformational preferences. This 25-residue long peptide in 20 mol % trifluoroethanol in water is in dynamic equilibrium between a helical and a more random conformation, and this equilibrium is shifted toward the more random structure as the temperature is raised. Part of the molecule, residues 10-18, exists in a stable helix at all temperatures studied (5, 25, and 50 degrees C). Propagation of the helix through the C-terminal end occurs at 25 degrees C, while the temperature must be lowered to 5 degrees C to observe any significant population of a helical conformation in the N-terminal region. These results argue that the Pro and Gly residues, which flank the helical segment, act to disfavor helix propagation on their N- or C-terminal sides, respectively. The influence of the Pro residue is stronger than that of the Gly. Furthermore, the most stable part of the helix in this signal peptide under the conditions studied is the hydrophobic core, which is the hallmark of functional signal sequences.

Design and characterization of a model alpha beta peptide.

CD and nmr spectroscopy were used to compare the conformational properties of two related peptides. One of the peptides, Model AB, was designed to adopt a helix-turn-extended strand (alpha beta) tertiary structure in water that might be stabilized by hydrophobic interactions between two leucine residues in the amino-terminal segment and two methionine residues in the carboxyl terminal segment. The other peptide, AB Helix, has the same amino acid sequence as Model AB except that it lacks the -Pro-Met-Thr-Met-Thr-Gly segment at the carboxyl-terminus. Although the carboxyl-terminal segment of Model AB was found to be unstructured, its presence increases the number of residues in a helical conformation, shifts the pKas of three ionizable side chains by 1 pH unit or more compared to an unstructured peptide, stabilizes the peptide as a monomer in high concentrations of ammonium sulfate, increases the conformational stability of residues at the terminal ends of the helix, and results in many slowly exchanging amide protons throughout the entire backbone of the peptide. These results suggest that interactions between adjacent segments in a small peptide can have significant structure organizing effects. Similar kinds of interactions may be important in determining the structure of early intermediates in protein folding and may be useful in the de novo design of independently folding peptides.

Side chain-backbone hydrogen bonding contributes to helix stability in peptides derived from an alpha-helical region of carboxypeptidase A.

Recently, Presta and Rose proposed that a necessary condition for helix formation is the presence of residues at the N- and C-termini (called NTBs and CTBs) whose side chains can form hydrogen bonds with the initial four amides and the last four carbonyls of the helix, which otherwise lack intrahelical hydrogen bonding partners. We have tested this hypothesis by conformational analysis by circular dichroism (CD) of a synthetic peptide corresponding to a region (171-188) of the protein carboxypeptidase A; in the protein, residues 174 to 186 are helical and are flanked by NTBs and CTBs. Since helix formation in this peptide may also be stabilized by electrostatic interactions, we have compared the helical content of the native peptide with that of several modified peptides designed to enable dissection of different contributions to helix stability. As expected, helix dipole interactions appear to contribute substantially, but we conclude that hydrogen bonding interactions as proposed by Presta and Rose also stabilize helix formation. To assist in comparison of different peptides, we have introduced two concentration-independent CD parameters which are sensitive probes of helix formation.

Impact of a micellar environment on the conformations of two cyclic pentapeptides.

In an effort to explore the influence of interfacial environments on reverse turns, we have performed a detailed analysis by nmr of the solution conformations of two cyclic pentapeptides in sodium dodecyl sulfate (SDS) micelles. The first peptide, cyclo (D-Phe1-Pro2-Gly3-D-Ala4-Pro5), adopts a single rigid conformation in solution (either chloroform or dimethylsulfoxide) and in crystals, whereas the second, cyclo (Gly1-Pro2-D-Phe3-Gly4-Val5), is much more flexible and adopts different conformations in the crystal and in solution. Both of these peptides are solubilized by SDS micelles, and nmr relaxation rates indicate that they are both partially immobilized by interaction with the micelles. Furthermore, some amide protons in both peptides participate in hydrogen bonds with water. In the presence of micelles, the former peptide retains a conformation essentially the same as that found in crystals and in solution, which consists of a beta turn and an inverse gamma turn. However, the micellar environment has a significant effect on the latter peptide. In particular, the population of a conformer containing a cis Gly-Pro peptide bond is increased significantly. The most likely conformation of the cis isomer, determined by a combination of nmr and restrained molecular dynamics, contains a Gly1-Pro2 delta turn and a gamma turn about D-Phe3. The nmr data on the trans isomer indicate that this isomer is averaging between two conformations that differ mainly in the orientation of the D-Phe3-Gly4 peptide bond.

Characterization of virtual coupling in the proton nuclear magnetic resonance spectrum of N,N'-diacetylchitobiose by two-dimensional J-resolved spectroscopy.

The anomeric proton of the reducing N-acetylglucosamine residue of beta-N,N'-diacetylchitobiose exhibited an unusual lineshape indicative of virtual coupling in the 250 MHz proton NMR spectrum. The two-dimensional J-resolved spectrum contained four lines associated with this proton, and this pattern can be used to distinguish virtual coupling from other effects. Anomeric protons of previously studied asparagine-linked glycopeptides which exhibited unusual lineshapes in the conventional NMR spectra did not exhibit the four line patterns indicative of virtual coupling in the two-dimensional J-resolved spectra. Although virtual coupling may partially account for the unusual lineshapes observed in the normal proton NMR spectra, the anomalous behavior observed in the two-dimensional J-resolved spectra of these glycopeptides cannot be explained solely by this phenomenon.

Cyclic pentapeptides as models for reverse turns: determination of the equilibrium distribution between type I and type II conformations of Pro-Asn and Pro-Ala beta-turns.

Cyclic pentapeptides are excellent models for reverse turns and have been used extensively in our laboratory to explore the influence of different amino acid sequences on turn preference. This paper is divided into two parts: In the first, we review our previous studies of cyclic pentapeptides. We summarize work that demonstrates the range of conformations possible within the cyclic pentapeptide backbone, the importance of sequence chirality in determining the backbone fold, and the utility of these cyclic pentapeptides as models for various turns. In the second, we present new results on two cyclic pentapeptides that contain beta-turns with Pro-Ala or Pro-Asn sequences in the i + 1 and i + 2 positions. By stereochemical criteria, a type I beta-turn is expected to be preferred by such L-L sequences. On the other hand, in proteins Asn occurs frequently in the i + 2 position of type II turns. We asked whether the same propensity would be manifest in an isolated model peptide, and if so, what the interactions were that influenced the relative stability of the type I and type II turns. To address these questions we have compared the conformational behavior of two peptides: cyclo(Gly-Pro-Ala-D-Phe-Pro) and cyclo(D-Ala-Pro-Asn-Gly-Pro). From previous studies, we anticipated that both peptides would contain an inverse gamma-turn and a beta-turn which consisted of either Gly-Pro-Ala-D-Phe or D-Ala-Pro-Asn-Gly in positions i to i + 3, respectively. Nuclear magnetic resonance analysis confirms this overall backbone conformation. Furthermore, quantitative nuclear Overhauser effect measurements in combination with molecular dynamics simulations and torsionally-forced energy minimizations have enabled us to determine that both type I and type II beta-turns are present in equilibrium in these peptides. The introduction of Asn in position i + 2 shifts this equilibrium significantly towards type II. We have done preliminary assessment of the possible side-chain/backbone conformations that contribute to the shift in populations.

Conformational behavior of Escherichia coli OmpA signal peptides in membrane mimetic environments.

Nuclear magnetic resonance and circular dichroism (CD) studies of isolated peptides corresponding to WT and mutant OmpA signal sequences are reported; all of the peptides adopt substantial amounts of alpha-helical structure both in 1:1 (v/v) trifluoroethanol (TFE)/water and in sodium dodecyl sulfate (SDS) micelles. In TFE/water, the helix begins after the positively charged N-terminal residues and is most stable in the hydrophobic core, which correlates with results obtained previously for other signal sequences. The helix is weaker between the hydrophobic core and the C-terminus; such a break in the helix appears to be common to other signal peptides studied previously and could be of functional importance. No clear correlation could be established between the helicity of the peptides in TFE/water and their in vivo activities. All the peptides have a higher alpha-helix content in SDS than in TFE/water, and there is a good correlation between helix content in SDS and in vivo activity. Helicity in SDS for the functional peptides increases both at the N-terminus and in the hydrophobic core, and is driven by a strong association of the core with the hydrophobic chains of the detergent. The extension of the helix toward the N-terminus may be a result of neutralization of the N-terminal positive charges by the headgroups of the micelles, which removes unfavorable electrostatic interactions with the helix dipole. All these comparisons were facilitated by the use of upfield shifts of H alpha protons in helical regions relative to random coil chemical shifts, which also yielded estimates of helical content that correlated well with the CD results.