Terminal sequence importance of de novo proteins from binary-patterned library: stable artificial proteins with 11- or 12-amino acid alphabet

Successful approaches of de novo protein design suggest a great potential to create novel structural folds and to understand natural rules of protein folding. For these purposes, smaller and simpler de novo proteins have been developed. Here, we constructed smaller proteins by removing the terminal sequences from stable de novo vTAJ proteins and compared stabilities between mutant and original proteins. vTAJ proteins were screened from an α3β3 binary-patterned library which was designed with polar/ nonpolar periodicities of α-helix and β-sheet. vTAJ proteins have the additional terminal sequences due to the method of constructing the genetically repeated library sequences. By removing the parts of the sequences, we successfully obtained the stable smaller de novo protein mutants with fewer amino acid alphabets than the originals. However, these mutants showed the differences on ANS binding properties and stabilities against denaturant and pH change. The terminal sequences, which were designed just as flexible linkers not as secondary structure units, sufficiently affected these physicochemical details. This study showed implications for adjusting protein stabilities by designing N- and C-terminal sequences.     

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.     

Folding simulations of three proteins having all α-helix,all β-strand and α/β-structures

We performed folding simulations of three proteins using four force fields, AMBER parm96, AMBER parm99, CHARMM 27 and OPLS-AA/L, in order to examine the features of these force fields. We studied three proteins, protein A (all α-helix), cold-shock protein (all β-strand) and protein G (α/β-structures), for the folding simulations. For the simulation, we used the simulated annealing molecular dynamics method, which was performed 50 times for each protein using the four force fields. The results showed that the secondary-structure-forming tendencies are largely different among the four force fields. AMBER parm96 favours β-bridge structures and extended β-strand structures, and AMBER parm99 favours α-helix structures and 3₁₀-helix structures. CHARMM 27 slightly favours α-helix structures, and there are also π-helix and β-bridge structures. OPLS-AA/L favours α-helix structures and 3₁₀-helix structures.     

Effect of pressure on the secondary structure of coiled coil peptide GCN4-p1

It has recently been demonstrated that pressure induces folding of the α-helix of an alanine-based peptide (AK20), which is a monomer in water (Imamura and Kato, Proteins 2009;76:911–918). The present study focused on a coiled coil peptide GCN4-p1, the α-helices of which associate via a hydrophobic core, to examine whether the pressure stability of the α-helices depends on the hydrophobic core. Fourier transform infrared spectroscopy was used to investigate the effect of pressure on the secondary structures of GCN4-p1. The infrared spectra of GCN4-p1 shows the two amide I' peaks at ∼ 1650 and ∼ 1630 cm^− 1 stemming from the solvent-inaccessible α-helix and the solvent-accessible α-helix, respectively. The intensities of both the peaks increase with increasing pressure, whereas they decrease with increasing temperature. This indicates that pressure induces both the α-helices of GCN4-p1 to fold. The present result suggests that the positive volume change upon unfolding of an α-helix is a common characteristic of peptides. The pressure-induced stabilization of the α-helices is discussed in comparison with the pressure denaturation of proteins.     

Protein design as a challenge for peptide chemists

All efforts to turn the ultimate goal in protein de novo design into reality–the construction of new macromolecules with predetermined three-dimensional structure and well-defined functionality–failed because the mechanism of folding has still to be unravelled. In the present review, various attempts to apply synthetic tools for inducing native-like structural features in peptides in order to bypass the folding problem are described. Besides well-established methods for the nucleation and stabilization of secondary structures, e.g. α-helices, β-sheets and β-turns, topological templates as ‘built-in’ folding devices have more recently become the key elements for the induction of protein-like folding units (template-assembled synthetic proteins, TASP). Progress in the synthetic strategy and structural characterization of this new type of macromolecules opens the way for the design of functional TASP molecules.     

Dehydrophenylalanine zippers: strong helix-helix clamping through a network of weak interactions

A decapeptide Boc-L-Ala-(Delta Delta Phe)(4)-L-Ala-(Delta Delta Phe)3-Gly-OMe (Peptide I) was synthesized to study the preferred screw sense of consecutive alpha,beta-dehydrophenylalanine (Delta Delta Phe) residues. Crystallographic and CD studies suggest that, despite the presence of two L-Ala residues in the sequence, the decapeptide does not have a preferred screw sense. The peptide crystallizes with two conformers per asymmetric unit, one of them a slightly distorted right-handed 3(10)-helix (X) and the other a left-handed 3(10)-helix (Y) with X and Y being antiparallel to each other. An unanticipated and interesting observation is that in the solid state, the two shape-complement molecules self-assemble and interact with an extensive network of C-H...O hydrogen bonds and pi-pi interactions, directed laterally to the helix axis with amazing regularity. Here, we present an atomic resolution picture of the weak interaction mediated mutual recognition of two secondary structural elements and its possible implication in understanding the specific folding of the hydrophobic core of globular proteins and exploitation in future work on de novo design.     

Structural model of the gas vesicle protein GvpA and analysis of GvpA mutants in vivo

Gas vesicles are gas-filled protein structures increasing the buoyancy of cells. The gas vesicle envelope is mainly constituted by the 8 kDa protein GvpA forming a wall with a water excluding inner surface. A structure of GvpA is not available; recent solid-state NMR results suggest a coil-α-β-β-α-coil fold. We obtained a first structural model of GvpA by high-performance de novo modelling. Attenuated total reflection (ATR)-Fourier transform infrared spectroscopy (FTIR) supported this structure. A dimer of GvpA was derived that could explain the formation of the protein monolayer in the gas vesicle wall. The hydrophobic inner surface is mainly constituted by anti-parallel β-strands. The proposed structure allows the pinpointing of contact sites that were mutated and tested for the ability to form gas vesicles in haloarchaea. Mutations in α-helix I and α-helix II, but also in the β-turn affected the gas vesicle formation, whereas other alterations had no effect. All mutants supported the structural features deduced from the model. The proposed GvpA dimers allow the formation of a monolayer protein wall, also consistent with protease treatments of isolated gas vesicles.     

Slow and Bimolecular Folding of a De Novo Designed Monomeric Protein DS119

De novo protein design offers a unique means to test and advance our understanding of how proteins fold. However, most current design methods are native structure eccentric and folding kinetics has rarely been considered in the design process. Here, we show that a de novo designed mini-protein DS119, which folds into a βαβ structure, exhibits unusually slow and concentration-dependent folding kinetics. For example, the folding time for 50 μM of DS119 was estimated to be ∼2 s. Stopped-flow fluorescence resonance energy transfer experiments further suggested that its folding was likely facilitated by a transient dimerization process. Taken together, these results highlight the need for consideration of the entire folding energy landscape in de novo protein design and provide evidence suggesting nonnative interactions can play a key role in protein folding.     

Identification of an “α-helix-extended segment-α-helix” conformation of the sixth transmembrane domain in DMT1

DMT1 (divalent metal ion transporter 1) is one member of a family of proton-coupled transporters that facilitate the cellular absorption of divalent metal ions. A pair of mutation-sensitive and highly conserved histidines in the sixth transmembrane domain (TM6) of DMT1 was found to be important for proton-metal ion cotransport. In the present work, we investigate the structures and locations of the peptides from TM6 of DMT1 and its H267A and H272A mutants in SDS micelles by CD and NMR methods. The circular dichroism studies show that the α-helix is a predominant conformation for the wildtype peptide and H267A mutant in SDS micelles, whereas the helicity is evidently decreased for H272A mutant. The pH value has little effect on the α-helical contents of the three peptides. The NMR studies indicate that the wildtype peptide in SDS micelles forms an “α-helix-extended segment-α-helix” structure in which the His267 locates near the central part of the extended segment, while the His272 is involved in the α-helical folding. Both histidines are buried in SDS micelles as evidenced by their pK_a values. The structure of the wildtype peptide is evidently changed by the mutations of H267A and H272A. The H267A mutant forms an ordered structure consisting of an α-helix from the C-terminus to the central part and continuous turns in the residual part. The extended structure in the central part of the wildtype peptide is abolished by H267A mutation. The H272A mutation mainly induces unfolding of the short helix in the N-terminal side, while the short helix in the C-terminal side and unordered conformation in the central part remain. All the three peptides are embedded in SDS micelles, and the H267A mutant is inserted more deeply due to increasing hydrophobicity in the central part of the peptide. The specific “α-helix-extended segment-α-helix” structure of TM6 may have an important implication for the binding of the transporter to H⁺ and metal ions and the conformation change induced by the mutations of two highly conserved histidines may be correlated to the deficiency of the transport activity of DMT1.     

Multiscale simulations of protein folding: application to formation of secondary structures

A multiscale simulation method of protein folding is proposed, using atomic representation of protein and solvent, combing genetic algorithms to determine the key protein structures from a global view, with molecular dynamic simulations to reveal the local folding pathways, thus providing an integrated landscape of protein folding. The method is found to be superior to previously investigated global search algorithms or dynamic simulations alone. For secondary structure formation of a selected peptide, RN24, the structures and dynamics produced by this method agree well with corresponding experimental results. Three most populated conformations are observed, including hairpin, β-sheet and α-helix. The energetic barriers separating these three structures are comparable to the kinetic energy of the atoms of the peptide, implying that the transition between these states can be easily triggered by kinetic perturbations, mainly through electrostatic interactions between charged atoms. Transitions between α-helix and β-sheet should jump over at least two energy barriers and may stay in the energetic trap of hairpin. It is proposed that the structure of proteins should be jointly governed by thermodynamic and dynamic factors; free energy is not the exclusive dominant for stability of proteins.     

The effects of codon usage on the formation of secondary structures of nucleocapsid protein of peste des petits ruminants virus

The nucleocapsid (N) protein of peste des petits ruminants virus (PPRV) with a conserved amino acid usage pattern plays an important role in viral replication. The primary objective of this study was to estimate roles of synonymous codon usages of PPRV N gene and tRNA abundances of host in the formation of secondary structure of N protein. The potential effects of synonymous codon usages of N gene and tRNA abundances of host on shaping different folding units (α-helix, β-strand and the coil) in N protein were estimated, based on the information about the modeling secondary structure of PPRV N protein. The synonymous codon usage bias was found in different folding units in PPRV N protein. To better understand the role of translation speed caused by variant tRNA abundances in shaping the specific folding unit in N protein, we modeled the changing trends of tRNA abundance at the transition boundaries from one folding unit to another folding unit (β-strand → coil, coil → β-strand, α-helix → coil, coil → α-helix). The obvious fluctuations of tRNA abundance were identified at the two transition boundaries (β-strand → coil and coil → β-strand) in PPRV N protein. Our findings suggested that viral synonymous codon usage bias and cellular tRNA abundance variation might have potential effects on the formation of secondary structure of PPRV N protein.     

Synthesis and β-sheet propensity of constrained N-amino peptides

The stabilization of β-sheet secondary structure through peptide backbone modification represents an attractive approach to protein mimicry. Here, we present strategies toward stable β-hairpin folds based on peptide strand N-amination. Novel pyrazolidinone and tetrahydropyridazinone dipeptide constraints were introduced via on-resin Mitsunobu cyclization between α-hydrazino acid residues and a serine or homoserine side chain. Acyclic and cyclic N-amino peptide building blocks were then evaluated for their effect on β-hairpin stability in water using a GB1-derived model system. Our results demonstrate the strong β-sheet stabilizing effect of the peptide N-amino substituent, and provide useful insights into the impact of covalent dipeptide constraint on β-sheet folding.     

De novo design of α,β-didehydrophenylalanine containing peptides: from models to applications

The de novo design of peptides and proteins has emerged as an approach for investigating protein structure and function. The success relies heavily on the ability to design relatively short peptides that can adopt stable secondary structures. To this end, substitution with α,β-dehydroamino acids, especially α,β-didehydrophenylalanine (ΔPhe or ΔF) has blossomed in manifold directions, providing a rich diversity of well-defined structural motifs. Introduction of α,β-didehydrophenylalanine induces β-bends in small and 3(10)-helices in longer peptide sequences. Most favorable conformation of ΔF residues are (φ,ψ) ～(60°, 30°), (-60°, -30°), (-60°, 150°), and (60°, -150°). These features have been exploited in designing helix-turn-helix, helical bundle arrangements, and glycine zipper type super secondary structural motifs. The unusual capability of α,β-didehydrophenylalanine ring to form a variety of multicentered interactions (N-H…O, C-H…O, C-H…π, and N-H…π) suggests its possible exploitation for future de novo design of supramolecular structures. This work has now been extended to the de novo design of peptides with antibiotic, antifibrillization activity, etc. More recently, self-assembling properties of small dehydropeptides have been explored. This review focuses primarily on the structural and functional behavior of α,β-didehydrophenylalanine containing peptides.     

The surface of β-sheet proteins contains amphiphilic regions which may provide clues about protein folding

A major bottleneck in the field of biochemistry is our limited understanding of the processes by which a protein folds into its native conformation. Much of the work on this issue has focused on the conserved core of the folded protein. However, one might imagine that a ubiquitous motif for unaided folding or for the recognition of chaperones may involve regions on the surface of the native structure. We explore this possibility by an analysis of the spatial distribution of regions with amphiphilic α-helical potential on the surface of β-sheet proteins. All proteins, Including β-sheet proteins, contain regions with amphiphilic α-helical potential. That is, any α-helix formed by that region would be amphiphilic, having both hydrophobic and hydrophilic surfaces. In the three-dimensional structure of all β-sheet proteins analyzed, we have found a distinct pattern in the spatial distribution of sequences with amphiphilic α-helical potential. The amphiphilic regions occur in ring shaped clusters approximately 20 to 30 Å in diameter on the surface of the protein. In addition, these regions have a strong preference for positively charged amino acids and a lower preference for residues not favorable to α-helix formation. Although the purpose of these amphiphilic regions which are not associated with naturally occurring α-helix is unknown, they may play a critical role in highly conserved processes such as protein folding. © 1996 Wiley-Liss, Inc.     

Influence of the lipid composition on the kinetics of concerted insertion and folding of melittin in bilayers

We have examined the kinetics of the adsorption of melittin, a secondary amphipathic peptide extracted from bee venom, on lipid membranes using three independent and complementary approaches. We probed (i) the change in the polarity of the ¹⁹Trp of the peptide upon binding, (ii) the insertion of this residue in the apolar core of the membrane, measuring the ¹⁹Trp-fluorescence quenching by bromine atoms attached on lipid acyl chains, and (iii) the folding of the peptide, by circular dichroism (CD). We report a tight coupling of the insertion of the peptide with its folding as an α-helix. For all the investigated membrane systems (cholesterol-containing, phosphoglycerol-containing, and pure phosphocholine bilayers), the decrease in the polarity of ¹⁹Trp was found to be significantly faster than the increase in the helical content of melittin. Therefore, from a kinetics point of view, the formation of the α-helix is a consequence of the insertion of melittin. The rate of melittin folding was found to be influenced by the lipid composition of the bilayer and we propose that this was achieved by the modulation of the kinetics of insertion. The study reports a clear example of the coupling existing between protein penetration and folding, an interconnection that must be considered in the general scheme of membrane protein folding.     

α-Helical assembly of biologically active peptides and designed helix bundle protein

The formation of α-helical assembly by complexing biologically active peptides with de novo designed protein is described. The de novo designed protein described here is a cystinelinked 4-helix bundle protein constructed with 80 amino acid residues and forms a hydrophobic core region surrounded by 4 helices in an aqueous solution. The biologically active peptides, such as melittin and human growth hormone releasing factor, contain the sequences that are able to form amphiphilic helices. These peptides alone do not form the α-helix structure in a diluted solution with low ion strength. But on mixing with the designed helix bundle protein, the peptides are strongly bound to the protein with the induction of α-helical structure in the biologically active peptides. The content of induced α-helix is in accord with that estimated from the amphiphilic sequence. The results mean that a novel architecture composed of α-helices is formed. Fluorescent and temperature-scanning measurement revealed that the α-helical assembly is constructed with hydrophobic interaction. Also, it is shown by means of fluorescence depolarization that the assembly has a compact globular form corresponding to 1 : 1 complex. © 1994 John Wiley & Sons, Inc.     

Dissection of the de novo designed peptide alpha t alpha: stability and properties of the intact molecule and its constituent helices

Alpha t alpha is a de novo designed 38-residue peptide [Fezoui et al. (1995) Protein Sci. 4, 286-295] that adopts a helical hairpin conformation in solution [Fezoui et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3675-3679; Fezoui et al. (1997) Protein Sci. 6, 1869-1877]. Since alpha t alpha was developed as a model system for protein folding at the stage where secondary structures interact and become mutually stabilizing, it is of interest to investigate the increase in stability that occurs with helix association. alpha t alpha was dissected into its component helices and the relative stabilities of the individual helices and the parent molecule were assessed. The Delta G0 of unfolding of alpha t alpha measured by guanidine hydrochloride denaturation was determined to be 3.4 kcal/mol. The equilibrium constant for folding of alpha t alpha was estimated from the Delta G0 as 338 and from hydrogen exchange measurements as 259. The stability of the helices in intact alpha t alpha over the individual helices increased by a factor of at least 37 based on amide proton exchange measurements. Sedimentation equilibrium studies showed very little association of the peptides to form either homo- or heterodimers, suggesting that helix association is stabilized by the high effective concentration of the helices caused by the presence of the connecting turn. The effects of salt and pH on the helicity of the component peptides are largely reflected in the intact molecule, implying that short-range interactions still make important contributions to the conformation of the intact molecule even though significant stabilization is caused by helix association.     

The effect of charge-charge interactions on the kinetics of alpha-helix formation

The formation of the monomeric α-helix represents one of the simplest scenarios in protein folding; however, our current understanding of the folding dynamics of the α-helix motif is mainly based on studies of alanine-rich model peptides. To examine the effect of peptide sequence on the folding kinetics of α-helices, we studied the relaxation kinetics of a 21-residue helical peptide, Conantokin-T (Con-T), using time-resolved infrared spectroscopy in conjunction with a laser-induced temperature jump technique. Con-T is a neuroactive peptide containing a large number of charged residues that is found in the venom of the piscivorous cone snail Conus tulipa. The temperature-jump relaxation kinetics of Con-T is distinctly slower than that of previously studied alanine-based peptides, suggesting that the folding time of α-helices is sequence-dependent. Furthermore, it appears that the slower folding of Con-T can be attributed to the fact that its helical conformation is stabilized by charge-charge interactions or salt bridges. Although this finding contradicts an earlier molecular dynamics simulation, it also has implications for existing models of protein folding.     

Slow and Bimolecular Folding of a De Novo Designed Monomeric Protein DS119

De novo protein design offers a unique means to test and advance our understanding of how proteins fold. However, most current design methods are native structure eccentric and folding kinetics has rarely been considered in the design process. Here, we show that a de novo designed mini-protein DS119, which folds into a βαβ structure, exhibits unusually slow and concentration-dependent folding kinetics. For example, the folding time for 50 μM of DS119 was estimated to be ∼2 s. Stopped-flow fluorescence resonance energy transfer experiments further suggested that its folding was likely facilitated by a transient dimerization process. Taken together, these results highlight the need for consideration of the entire folding energy landscape in de novo protein design and provide evidence suggesting nonnative interactions can play a key role in protein folding.     

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.