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.

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.

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.

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.

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.

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.

Design of a zinc-finger hydrolase with a synthetic αββ 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.

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.

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.

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.

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

The planar ribbon of the poly-L β-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.

Zinc-finger hydrolase: Computational selection of a linker and a sequence towards metal activation with a synthetic αββ 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 β-hairpin building blocks of an αββ 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.

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.

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.

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.

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.

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.

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.

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.

The interplay of sequence and stereochemistry in defining conformation in proteins and polypeptides.

Sequential specification of conformation in proteins and polypeptides is a triangular interplay involving the system of linked peptides, the sequences in side chains, and water as solvent. Stereochemistry in side chain linkages is obviously important in the interaction between all of the players, but no specification of its explicit role, if any, in linking sequence with conformation has been made. Flory and coworkers made a puzzling observation in 1967 that, when mutated from poly-L to alternating-L,D stereochemical structure, polypeptides will suffer a reduction in overall dimension or characteristic ratio by an astonishing factor of 10 and to a value even lower than that predicted for free rotation (Miller, W.

Computational design of proteins stereochemically optimized in size, stability, and folding speed.

Artificial proteins potentially barrier-free in the folding kinetics are approached computationally under the guidance of protein-folding theories. The smallest and fastest folding globular protein triple-helix-bundle (THB) is so modified as to minimize or eliminate its presumed barriers in folding speed. As the barriers may reside in the ordering of either secondary or tertiary structure, the elements of both secondary and tertiary structure in the protein are targeted for prenucleation with suitable stereochemically constrained amino acid residues.

The link between sequence and conformation in protein structures appears to be stereochemically established.

In search of the link between sequence and conformation in protein structures, we perform molecular dynamics analysis of the effect of stereochemical mutation in end-protected octa-alanine Ac-Ala8-NHMe from poly-L to an alternating-L,D structure. The mutation has a dramatic effect, transforming the peptide from a condition of extreme sensitivity to one of extreme insensitivity to solvent. Examining the molecular folds of poly-L and alternating-L,D structure in atomistic detail, we find them to differ in the relationship between peptide dipolar interactions at the local and nonlocal levels, either conflicting or harmonious depending upon the chain stereochemistry.

A small peptide stereochemically customized as a globular fold with a molecular cleft.

A boat shaped peptide molecular fold is generated by stereochemical modification of a 20-residue beta-hairpin peptide, making it a promising prototype for future optimization as a molecular receptor.

Simulated folding in polypeptides of diversified molecular tacticity: implications for protein folding and de novo design.

Stereochemistry could be a powerful variable for conformational tune up of polypeptides for de novo design. It may be also useful probe of possible role of interamide energetics in selection and stabilization of conformation. The homopolypeptides Ac-Xxx30-NHMe, with Xxx = Ala, Val, and Leu, of diversified stereochemical structure are generated by simulated racemization with a modified GROMOS-96 force field.