De novo design and characterization of a helical hairpin eicosapeptide; emergence of an anion receptor in the linker region

De novo design of supersecondary structures is expected to provide useful molecular frameworks for the incorporation of functional sites as in proteins. A 21 residue long, dehydrophenylalanine-containing peptide has been de novo designed and its crystal structure determined. The apolar peptide folds into a helical hairpin supersecondary structure with two right-handed helices, connected by a tetraglycine linker. The helices of the hairpin interact with each other through a combination of C-H.O and N-H.O hydrogen bonds. The folding of the apolar peptide has been realized without the help of either metal ions or disulphide bonds. A remarkable feature of the peptide is the unanticipated occurrence of an anion binding motif in the linker region, strikingly similar in conformation and function to the "nest" motif seen in several proteins. The observation supports the view for the possible emergence of rudimentary functions over short sequence stretches in the early peptides under prebiotic conditions.     

Design of a minimal protein oligomerization domain by a structural approach

Because of the simplicity and regularity of the alpha-helical coiled coil relative to other structural motifs, it can be conveniently used to clarify the molecular interactions responsible for protein folding and stability. Here we describe the de novo design and characterization of a two heptad-repeat peptide stabilized by a complex network of inter- and intrahelical salt bridges. Circular dichroism spectroscopy and analytical ultracentrifugation show that this peptide is highly alpha-helical and 100% dimeric tinder physiological buffer conditions. Interestingly, the peptide was shown to switch its oligomerization state from a dimer to a trimer upon increasing ionic strength. The correctness of the rational design principles used here is supported by details of the atomic structure of the peptide deduced from X-ray crystallography. The structure of the peptide shows that it is not a molten globule but assumes a unique, native-like conformation. This de novo peptide thus represents an attractive model system for the design of a molecular recognition system.     

The construction of new proteins: V. A template-assembled synthetic protein (TASP) containing both a 4-helix bundle and beta-barrel-like structure

The construction of a template-assembled synthetic protein (TASP) designed to contain both a 4-helix bundle and a beta-barrel as two folding "domains" is described. For the de novo design of proteins, amphiphilic helices (alpha) and beta-sheets (beta) are covalently attached to a template peptide (T) carrying functional side chains suitably oriented to promote intramolecular folding of the secondary structure blocks into a characteristic packing arrangement, i.e., T8-(4 alpha)(4 beta). The design of this new macromolecule was assisted by computer modeling, which suggested a low-energy conformation with tight hydrophobic packing of the secondary structure subunits. Solid-phase synthesis of the "two-domain" TASP molecule was achieved using orthogonal protection techniques. The solution properties as well as circular dichroism (CD) and infrared spectroscopy (IR) data under various experimental conditions are consistent with the folded conformation suggested by modeling.     

Rotational orientation of monomers within a designed homo-oligomer transmembrane helical bundle

A peptide designed to form a homo-oligomeric transmembrane helical bundle was reconstituted into lipid bilayers and studied by using (2)H NMR (nuclear magnetic resonance) with magic angle spinning to confirm that the helical interface corresponds to the interface intended in the design. The peptide belongs to a family of model peptides derived from a membrane-solubilized version of the water-soluble coiled-coil GCN4-P1. The variant studied here contains two asparagines thought to engage in interhelical hydrogen bonding critical to the formation of a stable trimer. For the NMR studies, three different peptides were synthesized, each with one of three consecutive leucines in the transmembrane region deuterium labeled. Prior to NMR data collection, polarized infrared spectroscopy was used to establish that the peptides were reconstituted in lipid bilayers in a transmembrane helical conformation. The (2)H NMR line shapes of the three different peptides are consistent with a trimer structure formed by the designed peptide that is stabilized by inter-helical hydrogen bonding of asparagines at positions 7 and 14.     

Design and characterization of a model αβ 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 (αβ) 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. © 1995 John Wiley & Sons, Inc.     

Alpha-helical antimicrobial peptides—Using a sequence template to guide structure-activity relationship studies

An important class of cytolytic antimicrobial peptides (AMPs) assumes an amphipathic, α-helical conformation that permits efficient interaction with biological membranes. Host defence peptides of this type are widespread in nature, and numerous synthetic model AMPs have been derived from these or designed de novo based on their characteristics. In this review we provide an overview of the ‘sequence template’ approach which we have used to design potent artificial helical AMPs, to guide structure-activity relationship studies aimed at their optimization, and to help identify novel natural AMP sequences. Combining this approach with the rational use of natural and non-proteinogenic amino acid building blocks has allowed us to probe the individual effects on the peptides' activity of structural and physico-chemical parameters such as the size, propensity for helical structuring, amphipathic hydrophobicity, cationicity, and hydrophobic or polar sector characteristics. These studies furthermore provided useful insights into alternative modes of action for natural membrane-active helical peptides.     

Dynamics of a de novo designed three-helix bundle protein studied by 15N, 13C, and 2H NMR relaxation methods

Understanding how the amino acid sequence of a polypeptide chain specifies a unique, functional three-dimensional structure remains an important goal, especially in the context of the emerging discipline of de novo protein design. Alpha3D is a single chain protein of 73 amino acids resulting from a de novo design effort. Previous solution nuclear magnetic resonance studies of alpha3D confirm that the protein adopts the designed structure of a three-helix bundle. Furthermore, alpha3D has been previously shown to possess all of the major thermodynamic and structural characteristics of natural proteins, though it shares no sequence homology to any protein sequence in the database. In this work, the backbone and side-chain dynamics of alpha3D were investigated using 15N, 13C, and 2H nuclear magnetic resonance relaxation methods with the aim of assessing the character of the internal motions of this native-like protein of de novo design. At the backbone level, both 15N and 13C(alpha) relaxation studies indicate highly restrictive motion on the picosecond to nanosecond time scale in the alpha-helical regions of alpha3D, with increasing mobility at the ends of the alpha-helices and in the two loop regions. This is largely consistent with what is seen in proteins of natural origin. Overall, the view provided by both 2H and 13C methyl relaxation methods suggest that the side chains of alpha3D are more dynamic compared to natural proteins. Regions of relative flexibility bound clusters of rigid methyl-bearing side-chain groups that are interspersed with aromatic and beta-branched amino acids. The time scale of motions associated with methyl-bearing side chains of alpha3D are significantly longer than that seen in natural proteins. These results indicate that the strategies underlying the design of alpha3D have largely, but not completely, captured both the structural and dynamic character of natural proteins.     

Conformational properties of five peptides corresponding to the entire sequence of glutathione transferase domain II

Five peptides matching the helices alpha4, alpha5, alpha6, alpha7, and alpha8, spanning the entire sequence of domain II of pG-STP1-1, have been synthesized and their conformations analyzed by far-UV CD spectroscopy. The results show that a5, a7, and a8 peptides are unstructured in water/2,2,2-trifluoroethanol (TFE) solutions. The a4-peptide also adopts random conformations in aqueous solvent. Moreover, the relative low helical content (20%), estimated for this peptide in the presence of 30% (v/v) TFE, suggests that the sequence of this protein fragment does not possess sufficient information for a strong helical propensity. On the contrary, the synthesized a6 peptide, in the presence of TFE, showed a relevant structural autonomy with a helical content (41%) which was significantly higher than that estimated, under the same conditions, for all other peptides. More in general in the presence of solvents less polar than water, the isolated a6 peptide shows the same helical conformation adopted by the corresponding alpha6-helix in the hydrophobic core of the protein. A n-capping box motif, strictly conserved at the N-terminal of the alpha6-helix of all GST and related protein including eucaryotic translation elongation factor (EF1gamma) and the yeast prion protein Ure2, plays an important role in the alpha-helix nucleation and stability of this protein fragment. The results suggest that the alpha6-helix might represent a nucleation site of GST folding and that the helical conformation of this region of the protein is an important requirement during earlier events of GST refolding.     

Ab initio prediction of the three-dimensional structure of a de novo designed protein: a double-blind case study

Ab initio structure prediction and de novo protein design are two problems at the forefront of research in the fields of structural biology and chemistry. The goal of ab initio structure prediction of proteins is to correctly characterize the 3D structure of a protein using only the amino acid sequence as input. De novo protein design involves the production of novel protein sequences that adopt a desired fold. In this work, the results of a double-blind study are presented in which a new ab initio method was successfully used to predict the 3D structure of a protein designed through an experimental approach using binary patterned combinatorial libraries of de novo sequences. The predicted structure, which was produced before the experimental structure was known and without consideration of the design goals, and the final NMR analysis both characterize this protein as a 4-helix bundle. The similarity of these structures is evidenced by both small RMSD values between the coordinates of the two structures and a detailed analysis of the helical packing.     

Stabilization of helical structure in two 17-residue amphipathic analogues of the C-terminal peptide of cytochrome C

The conformations of two 17-residue peptide analogues derived from the C-terminal sequence of pigeon cytochrome c (native sequence = KAERADLIAYLKQATAK) were examined in aqueous and lipid environments by CD spectroscopy. The two analogues, KKLLKKLIAYLKQATAK (K peptide) and EELLEELIAYLKQATAK (E peptide), were made amphipathic with respect to helical segregation by substituting a 6-residue sequence at the N-terminus of the native peptide. Their structures were compared to the native peptide under aqueous conditions of varying pH and temperature, and in the presence of liposomes composed of phosphatidylcholine and phosphatidylserine in the ratio of 9:1. The results indicated that the native peptide remains unstructured under all the conditions examined even though this region of the native molecule is surface exposed and helical. The E peptide, however, was helical under aqueous conditions at 25 degrees C from pH 2-10 with a maximum helicity at pH 4 (54% helix from analysis of CD data). The ellipticity of the E peptide at pH 4 and 8 was concentration dependent, indicating an aggregation phenomenon. In studies in which the CD spectrum was measured at different temperatures, the E peptide became more helical at lower temperatures at pH 4 but not at pH 8. Upon interaction with a lipid membrane in the form of liposomes, there appeared to be a slight destabilization in the structure of the E peptide. The K peptide in an aqueous environment behaved like the native peptide in that it was structureless at all pHs and temperatures examined. In the presence of liposomes, however, this peptide had a high helical content (75% helix from analysis of CD data). These findings suggest that while stabilization of the helix dipole with negative charges at the N-terminus are important in inducing helical conformation in the E peptide, hydrophobic interactions created during aggregation appear to provide the principal stabilizing force. The results with the K peptide demonstrate that the positive N-terminal sequence of this peptide is able to interact with the negatively charged head groups in the phospholipid membrane in such a fashion as to stabilize a helical structure that is not apparent in an aqueous environment alone.     

Analysis of peptide design in four-, five-, and six-helix bundle template assembled synthetic protein molecules

Four-, five-, and six-helix bundle template assembled synthetic proteins (TASPs) have been synthesized using disulfide bonds between cavitand templates and peptides, and characterized in terms of stability and structural specificity. The peptide sequence (CGGGEELLKKLEE LLKKG) used was originally designed for a four-helix bundle. The TASPs were analyzed using CD spectroscopy, chemical denaturation studies, NMR spectroscopy, sedimentation equilibria studies, and hydrophobic dye binding studies to determine the effect of a single peptide sequence when incorporated into bundles with different numbers of helices. If the design was indeed idealized for a four-helix bundle, then the five- and six-helix bundles should be less stable and manifest lower conformational specificity. The TASPs all demonstrated high stability and cooperative unfolding. However, the four-helix bundle was found to be significantly more stable and nativelike compared to the five- and six-helix bundles. This suggests that the peptide sequence is specific to the four-helix bundle, as designed. This result demonstrates the ability to design de novo proteins with specified structure, not just generic stability.     

Automated design of the surface positions of protein helices.

Using a protein design algorithm that quantitatively considers side-chain interactions, the design of surface residues of alpha helices was examined. Three scoring functions were tested: a hydrogen-bond potential, a hydrogen-bond potential in conjunction with a penalty for uncompensated burial of polar hydrogens, and a hydrogen-bond potential in combination with helix propensity. The solvent exposed residues of a homodimeric coiled coil based on GCN4-p1 were designed by using the Dead-End Elimination Theorem to find the optimal amino acid sequence for each scoring function. The corresponding peptides were synthesized and characterized by circular dichroism spectroscopy and size exclusion chromatography. The designed peptides were dimeric and nearly 100% helical at 1 degree C, with melting temperatures from 69-72 degrees C, over 12 degrees C higher than GCN4-p1, whereas a random hydrophilic sequence at the surface positions produced a peptide that melted at 15 degrees C. Analysis of the designed sequences suggests that helix propensity is the key factor in sequence design for surface helical positions.     

A structural and functional role for 11-mer repeats in alpha-synuclein and other exchangeable lipid binding proteins

We have used NMR spectroscopy and limited proteolysis to characterize the structural properties of the Parkinson's disease-related protein alpha-synuclein in lipid and detergent micelle environments. We show that the lipid or micelle surface-bound portion of the molecule adopts a continuously helical structure with a single break. Modeling alphaS as an ideal alpha-helix reveals a hydrophobic surface that winds around the helix axis in a right-handed fashion. This feature is typical of 11-mer repeat containing sequences that adopt right-handed coiled coil conformations. In order to bind a flat or convex lipid surface, however, an unbroken helical alphaS structure would need to adopt an unusual, slightly unwound, alpha11/3 helix conformation (three complete turns per 11 residues). The break we observe in the alphaS helix may allow the protein to avoid this unusual conformation by adopting two shorter stretches of typical alpha-helical structure. However, a quantitative analysis suggests the possibility that the alpha11/3 conformation may in fact exist in lipid-bound alphaS. We discuss how structural features of helical 11-mer repeats could play a role in the reversible lipid binding function of alpha-synuclein and generalize this argument to include the 11-mer repeat-containing apolipoproteins, which also require the ability to release readily from lipid surfaces. A search of protein sequence databases confirms that synuclein-like 11-mer repeats are present in other proteins that bind lipids reversibly and predicts such a role for a number of hypothetical proteins of unknown function.     

Sequence preference of α-helix N-terminal tetrapeptide

The α-helix is the most abundant secondary structure in proteins. Due to the specific i, i+4 hydrogen bond pattern, the two termini have unsatisfied hydrogen bonds, and are less constrained; in order to compensate for this, specific residues are preferred for the terminal positions. However, a naive combination of the statistically-preferred residues for each position may not result in a stable N-terminal helical sequence. In order to provide a set of preferable N-terminal peptides for α-helix design, we have studied the N-terminal tetrapeptide sequence motifs that are favorable for helix formation using statistical analysis and atomistic simulations. A set of tetrapeptide sequences including TEEE and TPEE were found to be favorable motifs. In addition to forming more hydrogen bonds in the helical conformation, the favorable motifs also tended to form more capping boxes. To empirically test our predictions, we obtained 10 peptides with different N-terminal motifs and measured their α-helical content by circular dichroism spectroscopy. The experimental results agreed qualitatively with the statistical and simulation results. Furthermore, some of the suggested preferable tetrapeptide sequences have been successfully applied in de novo protein design.     

Crumpled structure of the custom hydrophobic lytic peptide cecropin B3.

The solution structure of a custom lytic peptide, cecropin B3 (CB3), having two identical hydrophobic segments on both the N- and C-termini, was investigated by two-dimensional NMR spectroscopy. The need to determine the structure of this peptide is rooted in its specific ability to lyse lipid layers that have a high content of anionic lipid. The lytic activities of CB3 on cell membranes including cancer cells and bacteria is found to be less than cecropin B1. The results show that CB3 has four discrete segments forming alpha helical structures. The crumpled structure of CB3 provides evidence for the lysis of the lipid layer being via a pathway that differs from pore formation. The results in this study provide strong clues towards a rational design for a potent antimicrobial and antitumor peptide.     

Membrane-induced structure of the mammalian tachykinin neuropeptide gamma

Neuropeptide gamma (NPgamma) is a neurokinin-2 (NK-2) receptor selective agonist, which plays an important role in mediation of asthma and elicits a wide range of biological responses like bronchoconstriction, vasodepression and regulation of endocrine functions. The structure determination of this peptide agonist is important in understanding the molecular basis of peptide ligand recognition by the receptor and for rational drug design. In the present study we report the solution structure of NPgamma characterized by circular dichroism (CD) spectropolarimetry and 2D (1)H NMR spectroscopy in both aqueous and membrane mimetic solvents. Effect of calcium ions on the conformation of NPgamma was also studied using CD spectropolarimetry. Sequence-specific resonance assignments of protons have been made with the aid of correlation spectroscopy experiments and nuclear Overhauser effect spectroscopy experiments. The distance constraints obtained from the NMR data have been utilized to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that in water NPgamma prefers to be in an extended chain conformation whereas a helical conformation is induced in the central core and the C-terminal region of the peptide (K13-M21) in the presence of perdeuterated dodecylphosphocholine micelles, a membrane model system. A type II' beta turn from H9 to R11 precedes the helical core in the C-terminus of NPgamma. N-terminus of NPgamma also displays some degree of order and a possible turn structure. Conformation adopted by NPgamma in presence of lipid micelles represents a structural motif typical of NK-2 selective agonists and is similar to that observed for Neurokinin A in hydrophobic environment. The observed conformational features have been correlated to the binding ability and biological activity of NPgamma.     

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.     

Removing an interhelical salt bridge abolishes coiled-coil formation in a de novo designed peptide

Alpha-helical coiled coils represent a common protein oligomerization motif that are mainly stabilized by hydrophobic interactions occurring along their coiled-coil interface, the so-called hydrophobic seam. We have recently de novo designed and optimized a series of two-heptad repeat long coiled-coil peptides which are further stabilized by a complex network of inter- and intrahelical salt bridges. Here we have extended the de novo design of such two heptad-repeat long peptides by removing the central and most important g-e' Arg to Glu (g-e'RE) ionic interhelical interaction and replacing these residues by alanine residues. The effect of the missing interhelical ionic interaction on coiled-coil formation and stability has been analyzed by CD spectroscopy, analytical ultracentrifugation, and X-ray crystallography. We show that the peptide, while being highly alpha-helical, is no longer able to form a parallel coiled-coil structure but rather assumes an octameric globular helical assembly devoid of any coiled-coil interactions.     

De novo backbone and sequence design of an idealized alpha/beta-barrel protein: evidence of stable tertiary structure

We have designed, synthesized, and characterized a 216 amino acid residue sequence encoding a putative idealized alpha/beta-barrel protein. The design was elaborated in two steps. First, the idealized backbone was defined with geometric parameters representing our target fold: a central eight parallel-stranded beta-sheet surrounded by eight parallel alpha-helices, connected together with short structural turns on both sides of the barrel. An automated sequence selection algorithm, based on the dead-end elimination theorem, was used to find the optimal amino acid sequence fitting the target structure. A synthetic gene coding for the designed sequence was constructed and the recombinant artificial protein was expressed in bacteria, purified and characterized. Far-UV CD spectra with prominent bands at 222nm and 208nm revealed the presence of alpha-helix secondary structures (50%) in fairly good agreement with the model. A pronounced absorption band in the near-UV CD region, arising from immobilized aromatic side-chains, showed that the artificial protein is folded in solution. Chemical unfolding monitored by tryptophan fluorescence revealed a conformational stability (DeltaG(H2O)) of 35kJ/mol. Thermal unfolding monitored by near-UV CD revealed a cooperative transition with an apparent T(m) of 65 degrees C. Moreover, the artificial protein did not exhibit any affinity for the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (ANS), providing additional evidence that the artificial barrel is not in the molten globule state, contrary to previously designed artificial alpha/beta-barrels. Finally, 1H NMR spectra of the folded and unfolded proteins provided evidence for specific interactions in the folded protein. Taken together, the results indicate that the de novo designed alpha/beta-barrel protein adopts a stable three-dimensional structure in solution. These encouraging results show that de novo design of an idealized protein structure of more than 200 amino acid residues is now possible, from construction of a particular backbone conformation to determination of an amino acid sequence with an automated sequence selection algorithm.     

A novel, multilayer structure of a helical peptide.

X-ray diffraction analysis at 1.5 A resolution has confirmed the helical conformation of a de novo designed 18-residue peptide. However, the crystal structure reveals the formation of continuous molecular layers of parallel-packed amphiphilic helices as a result of much more extensive helix-helix interactions than predicted. The crystal packing arrangement, by virtue of distinct antiparallel packing interactions, segregates the polar and apolar surfaces of the helices into discrete and well-defined interfacial regions. An extensive "ridges-into-grooves" interdigitation characterizes the hydrophobic interface, whereas an extensive network of salt bridges and hydrogen bonds dominates the corresponding hydrophilic interface.