IDeAS: automated design tool for hetero-chiral protein folds.

Incorporating D amino acids in the protein design alphabet can in principle multiply the design space by many orders of magnitude. All native proteins are polymers composed of L chiral amino acids. Practically limitless in diversity over amino acid sequences, protein structure is limited in folds and thus shapes, principally due to the poly L stereochemistry of their backbone. To diversify shapes, we introduced both L- and D α-amino acids as design alphabets to explore the possibility of generating novel folds, varied in chemical as well as stereo-chemical sequence. Now, to have stereochemically-defined proteins tuned chemically, we present the Inverse Design and Automation Software, IDeAS. Retro-fitting side chains on a backbone with L and D stereochemistry, the software demonstrate functional fits over stereo-chemically diverse folds in a range of applications of interest in protein design.

Automated design evolution of stereochemically randomized protein foldamers.

Diversification of chain stereochemistry opens up the possibilities of an 'in principle' increase in the design space of proteins. This huge increase in the sequence and consequent structural variation is aimed at the generation of smart materials. To diversify protein structure stereochemically, we introduced L- and D-α-amino acids as the design alphabet. With a sequence design algorithm, we explored the usage of specific variables such as chirality and the sequence of this alphabet in independent steps. With molecular dynamics, we folded stereochemically diverse homopolypeptides and evaluated their 'fitness' for possible design as protein-like foldamers. We propose a fitness function to prune the most optimal fold among 1000 structures simulated with an automated repetitive simulated annealing molecular dynamics (AR-SAMD) approach. The highly scored poly-leucine fold with sequence lengths of 24 and 30 amino acids were later sequence-optimized using a Dead End Elimination cum Monte Carlo based optimization tool. This paper demonstrates a novel approach for the de novo design of protein-like foldamers.

N-terminal diproline and charge group effects on the stabilization of helical conformation in alanine-based short peptides: CD studies with water and methanol as solvent.

Protein folding problem remains a formidable challenge as main chain, side chain and solvent interactions remain entangled and have been difficult to resolve. Alanine-based short peptides are promising models to dissect protein folding initiation and propagation structurally as well as energetically. The effect of N-terminal diproline and charged side chains is assessed on the stabilization of helical conformation in alanine-based short peptides using circular dichroism (CD) with water and methanol as solvent. A1 (Ac-Pro-Pro-Ala-Lys-Ala-Lys-Ala-Lys-Ala-NH2 ) is designed to assess the effect of N-terminal homochiral diproline and lysine side chains to induce helical conformation. A2 (Ac-Pro-Pro-Glu-Glu-Ala-Ala-Lys-Lys-Ala-NH2 ) and A3 (Ac-dPro-Pro-Glu-Glu-Ala-Ala-Lys-Lys-Ala-NH2 ) with N-terminal homochiral and heterochiral diproline, respectively, are designed to assess the effect of Glu...Lys (i, i + 4) salt bridge interactions on the stabilization of helical conformation. The CD spectra of A1, A2 and A3 in water manifest different amplitudes of the observed polyproline II (PPII) signals, which indicate different conformational distributions of the polypeptide structure. The strong effect of solvent substitution from water to methanol is observed for the peptides, and CD spectra in methanol evidence A2 and A3 as helical folds. Temperature-dependent CD spectra of A1 and A2 in water depict an isodichroic point reflecting coexistence of two conformations, PPII and ß-strand conformation, which is consistent with the previous studies. The results illuminate the effect of N-terminal diproline and charged side chains in dictating the preferences for extended-ß, semi-extended PPII and helical conformation in alanine-based short peptides. The results of the present study will enhance our understanding on stabilization of helical conformation in short peptides and hence aid in the design of novel peptides with helical structures. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Scrutiny of chain-length and N-terminal effects in α-helix folding: a molecular dynamics study on polyalanine peptides.

Protein folding remains an unsolved problem as main-chain, side-chain, and solvent interactions remain entangled and have been hard to resolve. Polyalanines are promising models to analyze protein folding initiation and propagation structurally as well as energetically. In the present work, the effect of chain-length and N-terminal residue stereochemistry in polyalanine peptides are investigated for their role in the nucleation of α-helical conformation. The end-protected polyalanine peptides, tetra-alanine, Ac-LAla4-NHMe (Ia) and Ac-DAla-LAla3-NHMe (Ib), hexa-alanine, Ac-LAla6-NHMe (IIa) and Ac-DAla-LAla5-NHMe (IIb), and octa-alanine, Ac-LAla8-NHMe (IIIa) and Ac-DAla-LAla7-NHMe (IIIb), are assessed as chain-length and stereochemical-structure perturbed models. The appreciable variations in the sampling of α-helical conformation, including a sampling of α-helix folds, due to the cooperative effect of chain-length and N-terminal residue stereochemistry have been noted. The electrostatics of α-helical conformation rather than the conformational entropy of the main-chain appear to be decisive in the initiation of α-helix folding. The results of the present work will enhance our understanding on the nucleation of α-helical conformation in short peptides and aid in the design of novel peptides with α-helical structure that can modulate disease-related protein-protein interactions.

Automated protein design: Landmarks and operational principles.

Protein design has an eventful history spanning over three decades, with handful of success stories reported, and numerous failures not reported. Design practices have benefited tremendously from improvements in computer hardware and advances in scientific algorithms. Though protein folding problem still remains unsolved, the possibility of having multiple sequence solutions for a single fold makes protein design a more tractable problem than protein folding. One of the most significant advancement in this area is the implementation of automated design algorithms on pre-defined templates or completely new folds, optimized through deterministic and heuristic search algorithms. This progress report provides a succinct presentation of important landmarks in automated design attempts, followed by brief account of operational principles in automated design methods.

Algorithm to design inhibitors using stereochemically mixed l,d polypeptides: Validation against HIV protease.

Polypeptides have potential to be designed as drugs or inhibitors against the desired targets. In polypeptides, every chiral α-amino acid has enantiomeric structural possibility to become l or d amino acids and can be used as design monomer. Among the various possibilities, use of stereochemistry as a design tool has potential to determine both functional specificity and metabolic stability of the designed polypeptides. The polypeptides with mixed l,d amino acids are a class of peptidomimitics, an attractive drug like molecules and also less susceptible to proteolytic activities. Therefore in this study, a three step algorithm is proposed to design the polypeptides against desired drug targets. For this, all possible configurational isomers of mixed l,d polyleucine (Ac-Leu8-NHMe) structure were randomly modeled with simulated annealing molecular dynamics and the resultant library of discrete folds were scored against HIV protease as a model target. The best scored folds of mixed l,d structures were inverse optimized for sequences in situ and the resultant sequences as inhibitors were validated for conformational integrity using molecular dynamics. This study presents and validates an algorithm to design polypeptides of mixed l,d structures as drugs/inhibitors by inverse fitting them as molecular ligands against desired target.

Design of a zinc-finger hydrolase with a synthetic αßß protein.

Recent advances in protein design have opened avenues for the creation of artificial enzymes needed for biotechnological and pharmaceutical applications. However, designing efficient enzymes remains an unrealized ambition, as the design must incorporate a catalytic apparatus specific for the desired reaction. Here we present a de novo design approach to evolve a minimal carbonic anhydrase mimic. We followed a step-by-step design of first folding the main chain followed by sequence variation for substrate binding and catalysis. To optimize the fold, we designed an αßß protein based on a Zn-finger. We then inverse-designed the sequences to provide stability to the fold along with flexibility of linker regions to optimize Zn binding and substrate hydrolysis. The resultant peptides were synthesized and assessed for Zn and substrate binding affinity by fluorescence and ITC followed by evaluation of catalytic efficiency with UV-based enzyme kinetic assays. We were successful in mimicking carbonic anhydrase activity in a peptide of twenty two residues, using p-nitrophenyl acetate as a CO2 surrogate. Although our design had modest activity, being a simple structure is an advantage for further improvement in efficiency. Our approach opens a way forward to evolving an efficient biocatalyst for any industrial reaction of interest.

Redox specificity of 2-hydroxyacid-coupled NAD(+)/NADH dehydrogenases: a study exploiting "reactive" arginine as a reporter of protein electrostatics.

With "reactive" arginine as a kinetic reporter, 2-hydroxyacid dehydrogenases are assessed in basis of their specialization as NAD(+)-reducing or NADH-oxidizing enzymes. Specifically, M4 and H4 lactate dehydrogenases (LDHs) and cytoplasmic and mitochondrial malate dehydrogenases (MDHs) are compared to assess if their coenzyme specificity may involve electrostatics of cationic or neutral nicotinamide structure as the basis. The enzymes from diverse eukaryote and prokaryote sources thus are assessed in "reactivity" of functionally-critical arginine as a function of salt concentration and pH. Electrostatic calculations were performed on "reactive" arginines and found good correspondence with experiment. The reductive and oxidative LDHs and MDHs are assessed in their count over ionizable residues and in placement details of the residues in their structures as proteins. The variants found to be high or low in ΔpKa of "reactive" arginine are found to be also strong or weak cations that preferentially oxidize NADH (neutral nicotinamide structure) or reduce NAD(+) (cationic nicotinamide structure). The ionized groups of protein structure may thus be important to redox specificity of the enzyme on basis of electrostatic preference for the oxidized (cationic nicotinamide) or reduced (neutral nicotinamide) coenzyme. Detailed comparisons of isozymes establish that the residues contributing in their redox specificity are scrambled in structure of the reductive enzyme.

Aromatic interactions at atom-to-atom contact and just beyond: a case study of protein interactions of NAD⁺/NADP⁺.

We probed aromatic-protein interactions based on specificity of enrichment of protein residues across a contact-based cutoff. Thus, 155 protein-NAD(+)/NADP(+) complexes were analyzed for enrichments within 10Å of centroids of aromatic groups of the ligand when the residues were contacted and not contacted with the aromatic ligand. Specifically, neutral-adenine and cationic-nicotinamide groups of the oxidized coenzymes evoked interest to know whether the contrast of charge or the shared aromaticity will manifest in the enrichments across the cutoff. We found that when in contact, the enrichments are highly specific for nicotinamide and adenine-aromatic structures, and thus possibly complex in the basis, but when not in contact, they are generic for charge and aromaticity of the structures, and thus possibly specific in the basis. The order of enrichments over the contacted residues is Tyr>Cys>Thr>His>Asn>Ser>Met>Ile>Phe against nicotinamide-π(+) structure and Asp>Ile>Thr>His>Arg>Tyr>Gly>Val against adenine-π structure, while the order over the non-contacted residues is Trp>Gly>His>Asn>Cys>Met>Tyr>Ser>Thr>Phe against nicotinamide-π(+) structure and Asn>Thr>Ser>Gly>Cys>His>Val against adenine-π structure. Neutral Trp, His, Tyr, and Phe, but not cationic Arg, are thus the non-contacted residues enriched specifically against nicotinamide-π(+) structure, while Asn, Gly, Thr, Ser, and Cys are the non-contacted residues enriched generically against both the nicotinamide-π(+) and adenine-π aromatic structures. By analyzing the enriched groups in their geometric specificities, we found that, the enrichments against nicotinamide cation manifest the specificity expected of cation-π interaction and against nicotinamide- and adenine-aromatic groups manifest the specificity expected of dipole-π interaction. The cutoff-based method is proven valuable in probing protein-ligand interactions in the physics involved.

Stereochemistry and solvent role in protein folding: nuclear magnetic resonance and molecular dynamics studies of poly-L and alternating-L,D homopolypeptides in dimethyl sulfoxide.

The competing interactions folding and unfolding protein structure remain obscure. Using homopolypeptides, we ask if poly-L structure may have a role. We mutate the structure to alternating-L,D stereochemistry and substitute water as the fold-promoting solvent with methanol and dimethyl sulfoxide (DMSO) as the fold-denaturing solvents. Circular dichroism and molecular dynamics established previously that, while both isomers were folded in water, the poly-L isomer was unfolded and alternating-L,D isomer folded in methanol. Nuclear magnetic resonance and molecular dynamics establish now that both isomers are unfolded in DMSO. We calculated energetics of folding-unfolding equilibrium with water and methanol as solvents. We have now calculated interactions of unfolded polypeptide structures with DMSO as solvent. Methanol was found to unfold and water fold poly-L structure as a dielectric. DMSO has now been found to unfold both poly-L and alternating-L,D structures by strong solvation of peptides to disrupt their hydrogen bonds. Accordingly, we propose that while linked peptides fold protein structure with hydrogen bonds they unfold the structure electrostatically due to the stereochemical effect of the poly-L structure. Protein folding to ordering of peptide hydrogen bonds with water as canonical solvent may thus involve two specific and independent solvent effects-one, strong screening of electrostatics of poly-L linked peptides, and two, weak dipolar solvation of peptides. Correspondingly, protein denaturation may involve two independent solvent effects-one, weak dielectric to unfold poly-L structure electrostatically, and two, strong polarity to disrupt peptide hydrogen bonds by solvation of peptides.

Cured of "stickiness", poly-L ß-hairpin is promoted with LL-to-DD mutation as a protein and a hydrolase mimic.

The planar ribbon of the poly-L ß-hairpin is modified to a local ~90° bend by mutating a cross-strand pair of residues from LL to DD structure. The bend is furnished aromatic side chains in proximity of acid-base-nucleophile side chains, toward the possibility of catalyzed hydrolysis of an active-site-anchored substrate. Six sequences permuted in putative catalytic side chains are evaluated for activity and variability as hydrolase enzymes. Studies using CD, NMR, spectorofluorometry, ITC, and molecular dynamics establish that the sequences over the bent ß-hairpin are by and large aggregation-free folds soluble to at least millimolar concentration, and thus remarkably contrasted with "stickiness" of the canonical poly-L ß-hairpin. The heterochiral fold displays cooperative ordering and affinity for acetylcholine, p-nitrophenylacetate, and p-nitrophenylphosphate, presumably as the ligands in its aromatic pocket as a bent hairpin. The fold displays hydrolytic activity against p-nitrophenylacetate and manifests saturation kinetics with respect to substrate concentration. However, the catalysis power is feeble, which remains unaffected by repositioning acid-base-nucleophile side chains. Stereochemistry is proven to be critical in the balance between mutually competitive forces of polypeptide structure involved in guidance of folding or aggregation of the structure. Residue stereochemistry is confirmed in its value as the alphabet for design of protein folds to desired molecular shapes.

Zinc-finger hydrolase: Computational selection of a linker and a sequence towards metal activation with a synthetic αßß protein.

The zinc-finger protein is targeted for computational redesign as a hydrolase enzyme. Successful in having zinc activated for hydrolase function, the study validates the stepwise approach to having the protein tuned in main-chain structure stereochemically and over side chains chemically. A leucine homopolypeptide, harboring histidines to tri coordinate zinc and d-amino-acid-nucleated α-helix and ß-hairpin building blocks of an αßß protein, is taken up for modeling, first with cyana, in a mixed-chirality linker between the building blocks, and then with IDeAS, in a sequence over side chains. The designed mixed-chirality polypeptide structure is proven to order as an intended αßß fold and capture zinc to activate its role as a hydrolase catalyst. The design approach to have protein folds defined stereochemically and receptor and catalysis functions defined chemically is presented, and illustrates L- and D-α-amino-acid structures as the alphabet integrating chemical- and stereochemical-structure variables as its letters.

Protein homomers in point-group assembly: symmetry making and breaking are specific and distinctive in their codes of chemical alphabet in side chains.

Oligomerizing to point-group symmetry, protein oligomers need to have the symmetry broken for biologically crucial functions, such as, allosteric regulation, enzyme catalysis, and so forth. In the making of symmetry, based on self assembly, and the breaking of symmetry, based on intermolecular interactions, proteins may manifest, like their other functions, specific scripts over the coding alphabet in side chains. To address the possibility, we analyzed 82 protein homodimers in their C(2)-symmetry-related side chains across noncrystallographic interfaces, to know if they may be identical or distinct in conformation, and thus conserved or broken in symmetry. We find the propensity to conformational mismatch across interfaces correlated with side-chain chemical structure, low to very low in aromatic Trp, Tyr, His, Phe, and Arg, and high to very high in aliphatic Val, Pro, Met, Glu, Ser, Lys, Gln, Asn, and Asp, related not to polarity but, interestingly, to aromaticity of the structure. The organizational plan having aromatics embedded in a hub of aliphatic-nonpolar groups and a surrounding rim of aliphatic-polar groups, called "hotspot," has been known to direct protein-protein interaction. Finding conformational-mismatch propensities of side chains congruous with their specific chemical roles in protein-protein interaction, we propose that aromatic side chains will drive protein homomers to high symmetry, while polar- and nonpolar aliphatic side chains will drive them to the functionally-necessitated breaks of symmetry. Side chains are in their roles as protein-coding alphabet illuminated in the physics, which is discussed.

Homochiral stereochemistry: the missing link of structure to energetics in protein folding.

The notion is tested that homochiral stereochemistry being ubiquitous to protein structure could be critical to protein folding as well, causing it to become frustrated energetically providing the basis for its solvent- and sequence-mediated control. The proof in support of the notion is found in a consensus of experiment and computation according to which suitable oligopeptides are in their folding-unfolding equilibria, at both macrostate and microstate levels, susceptible to dielectric because of the conflict of peptide-chain electrostatics with interpeptide hydrogen bonds when the structure is poly-L but not when it is alternating-L,D. The argument is thus made that homochiral stereochemistry may in protein folding provide the unifying basis for its solvent- and sequence-mediated control based on screening of peptide-chain electrostatics under conflict with folding of the chain due to homochiral stereochemistry. Dielectric is brought into spotlight as the effect comparatively obscure but presumably critical to the folding in protein structure for its control.

Electrostatics-defying interaction between arginine termini as a thermodynamic driving force in protein-protein interaction.

Apparent electrostatics-defying clustering of arginines attributed as screening effect of solvent is in this study examined as a possible thermodynamic driving force in protein-protein interaction. A dataset of 266 protein dimers is found to have approximately 22% arginines mutually paired and approximately 17% pairs in interaction across interfaces and thus putative "hotspots" of protein-protein interaction. The pairing, uncorrelated with inter or intramolecular context, could be contributing in protein folding as well, and, uncorrelated with solvent access, could be driven by effects that are generic to solvent and protein structures. Mutually stacked at shorter distances but in diverse geometrical modes otherwise, the cations tend to be in gross deficit of hydrogen-bond partners, and contributing electrostatics across protein-protein interface that, on average, is repulsive for protein-protein interaction. Embedded in local environment enriched in polarizable residues, aromatic, aliphatic, and anionic, the arginines may contribute to protein-protein interaction via environmental polarization response to electrostatics of cation clustering, a possible new principle in molecular recognition.

Protein design with L- and D-alpha-amino acid structures as the alphabet.

Summarizing the implications of homochiral structures in interpeptide interactions, not only in the topology but also possibly in the physics of protein folding, this Account provides an overview of the concept of shape-specific protein design using D- and L-(alpha)amino acid structures as the alphabet. The molecular shapes accessible in de novo protein design are stereochemically defined. Indeed, the defining consideration for shape specificity in proteins to be alpha-helix/beta-sheet composites is the L configuration of the alpha-amino acid structures. The stereospecificity in shapes implies that protein shapes may be diversifiable stereochemically, that is, designable de novo, using D and L structures as the alphabet. Indeed, augmented with D enantiomers, Nature's alphabet will expand greatly in the diversity of polypeptide stereoisomers, for example, from 1(30) to 2(30)--that is, from one to ca. one billion--for a modestly sized 30-residue polypeptide. Furthermore, with each isomer having conformers stereospecific to its structure, molecular folds of specific shapes may be approachable sequentially when D and L structures are used as the alphabet. Illustrating the promise, 14-20-residue bracelet-, boat-, canoe-, and cup-shaped molecular folds were designed stereochemically or implemented as specific sequence plans in the D- and L-alpha-amino acid alphabet. In practical terms, canonical poly-L peptide folds were modified to the desired shapes via stereochemical mutations invoking enantiomer symmetries in the Ramachandran phi,psi space as the logic. For example, in designing the boat-shaped fold, the canonical beta-hairpin was reengineered in its flat planar structure via multiple coordinated L-to-D mutations in its position specific cross-strand neighbor residues, upturning its ends enclosing six side chains in a molecular cleft. While affirming the generality of the approach, the 20-residue molecular canoe and the 14-residue molecular cup are also presented as examples of the scope of functional design. The canoe, possessing alkali cation-specific catgrips in its main chain, and the cup, featuring an organic cation-specific aromatic triad in its side chains, do indeed display desired specificities in their ligand binding. Stereochemistry is, therefore, the crucial specifier of protein shapes and valuable as the tool for shape-specific protein design. Proteins in general, whether poly-L or mixed-D,L, require sequence effects of amino acid side chain structures for their stability, if not also for specifying them conformationally. The principles underlying these phenomena remain a puzzle, but studies invoking a stereochemical mutation approach to the problem have suggested that the poly-L structure may be crucial to the principles of sequential encoding of protein structures in amino acid side chains as the alphabet.

Analysis of the structural consensus of the zinc coordination centers of metalloprotein structures.

In a recent sequence-analysis study it was concluded that up to 10% of the human proteome could be comprised of zinc proteins, quite varied in the functional spread. The native structures of only few of the proteins are actually established. The elucidation of rest of the sequences of not just human but even other actively investigated genomes may benefit from knowledge of the structural consensus of the zinc-binding centers of the currently known zinc proteins. Nearly four hundred X-ray and NMR structures in the database of zinc-protein structures available as of April 2007 were investigated for geometry and conformation in the zinc-binding centers; separately for the structural and catalytic proteins and individually in the zinc centers coordinated to three and four amino-acid ligands. Enhanced cysteine involvement in agreement with the observation in human proteome has been detected in contrast with previous reports. Deviations from ideal coordination geometries are detected, possible underlying reasons are investigated, and correlations of geometry and conformation in zinc-coordination centers with protein function are established, providing possible benchmarks for putative zinc-binding patterns of the burgeoning genome data.

A mixed-alpha,beta miniprotein stereochemically reprogrammed to high-binding affinity for acetylcholine.

The protein-structure space is limited to L configuration in the asymmetric alpha-amino acid structures; the function space, on other hand, seems limitless because of the chemical diversity in the amino acid side chain structures. The chemical diversity in side chain structure may be multiplied beneficially with the stereochemical diversity in main chain structure; thus, de novo protein design may be explored for customizing molecular structures stereochemically and molecular functions chemically. Illustrating de novo design in the structure space of L and D alphabet, canonical all-beta folds of poly-L structure were reprogrammed as bracelet, boat, and canoe-shaped molecules-the "boat" as a receptor-like pocket and the "canoe" as a metal-ion receptor-simply by mutating specific L-amino acid residues to the corresponding D stereochemical structure. Demonstrating customization of molecular shape stereochemically and function chemically, a 15-residue mixed-alpha, beta miniprotein of canonical poly-L structure is now reprogrammed stereochemically as a cup-shaped receptor for acetylcholine via cation-pi interaction with a triad of aromatic side chains placed in mimicry of the acetylcholine-receptor sites both natural and artificial. Evidence from CD, fluorescence, NMR, DSC, ITC, MD, and molecular-docking studies is presented to show that a rationally designed 15-residue mixed-L, D peptide is a cooperatively ordered molecular fold in the stereochemically specified molecular morphology, submicromolar in affinity of acetylcholine and thus an acetylcholine receptor exceptionally small and simple. .

A double catgrip mixed L and D mini protein only 20 residues long.

Stereochemistry limits but also defines proteins, as conformational constructs stereospecific for poly-L structure. Employed as a variable in sequence, stereochemistry could make proteins customizable in the letters of L and D amino acid alphabet. In proof of concept, we previously demonstrated stereochemical reengineering of canonical beta-hairpins as bracelet and boat shaped molecules. Illustrating the prospect for functional design, a 20-residue four-stranded mini-beta protein is now customized stereochemically as a canoe shaped molecule. A conformational construct of four side by side hydrogen-bonded strands in alternately (l)beta, (d)beta conformation, joined via Type-II/II' beta-turns, is planned to be preponderantly apolar in beta-sheet favoring residues, interspersing two ion pairs, and suitably L and D in sequence. Synthesis followed by MD, NMR, CD, and MALDI-MS studies established the molecule as a canoe shaped fold in water, demonstrable in affinity of alkali and alkaline-earth metal ions as expected given its catgrip like elements. Another success in accomplishing a synthetic miniprotein complex in stereochemistry and stereospecific in conformation, exceptionally small yet functional in metal ion affinity, affirms the value in combined L and D alphabet in programming molecular shapes and functions stereochemically.