Chemical protein synthesis: Inventing synthetic methods to decipher how proteins work.

Total chemical synthesis of proteins has been rendered practical by the chemical ligation principle: chemoselective condensation of unprotected peptide segments equipped with unique, mutually reactive functional groups, enabled by formation of a non-native replacement for the peptide bond. Ligation chemistries are briefly described, including native chemical ligation - thioester-mediated, amide-forming reaction at Xaa-Cys sites - and its extensions. Case studies from the author's own works are used to illustrate the utility and applications of chemical protein synthesis. Selected recent developments in the field are briefly discussed.

Protein conformational dynamics in the mechanism of HIV-1 protease catalysis.

We have used chemical protein synthesis and advanced physical methods to probe dynamics-function correlations for the HIV-1 protease, an enzyme that has received considerable attention as a target for the treatment of AIDS. Chemical synthesis was used to prepare a series of unique analogues of the HIV-1 protease in which the flexibility of the "flap" structures (residues 37-61 in each monomer of the homodimeric protein molecule) was systematically varied. These analogue enzymes were further studied by X-ray crystallography, NMR relaxation, and pulse-EPR methods, in conjunction with molecular dynamics simulations. We show that conformational isomerization in the flaps is correlated with structural reorganization of residues in the active site, and that it is preorganization of the active site that is a rate-limiting factor in catalysis.

Use of model peptide reactions for the characterization of kinetically controlled ligation.

Since the introduction of kinetically controlled ligation (KCL), a chemoselective reaction between a peptide-(α)thioarylester and a Cys-peptide-(α)thioalkylester, KCL has been utilized for the total chemical synthesis of large proteins (i.e., lysozyme and HIV-protease) by providing fully convergent synthetic routes. Although KCL has the potential to become an important chemistry for protein synthesis, the principle of KCL is not fully characterized. In particular, prior work on KCL has focused on the reactivity difference of the two different -(α)thioester forms-alkyl vs aryl. Another equally important feature of KCL, Xaa-Cys ligation sites, has not been investigated. The work reported here describes combinatorial KCL reactions using model peptides to dissect the interplay of the Xaa(1), Xaa(2), -(α)thioarylester, and -(α)thioalkylester. Results from these studies provide fundamental insights into the KCL reaction, and will lead to the optimal synthetic route for the routine chemical synthesis of large target protein molecules.

Total chemical synthesis of a D-enzyme: the enantiomers of HIV-1 protease show reciprocal chiral substrate specificity [corrected].

The D and L forms of the enzyme HIV-1 protease have been prepared by total chemical synthesis. The two proteins had identical covalent structures. However, the folded protein-enzyme enantiomers showed reciprocal chiral specificity on peptide substrates. That is, each enzyme enantiomer cut only the corresponding substrate enantiomer. Reciprocal chiral specificity was also evident in the effect of enantiomeric inhibitors. These data imply that the folded forms of the chemically synthesized D- and L-enzyme molecules are mirror images of one another in all elements of the three-dimensional structure. Enantiomeric proteins are expected to display reciprocal chiral specificity in all aspects of their biochemical interactions.

Constructing proteins by dovetailing unprotected synthetic peptides: backbone-engineered HIV protease.

Backbone-engineered HIV-1 protease was prepared by a total chemical synthesis approach that combines the act of joining two peptides with the generation of an analog structure. Unprotected synthetic peptide segments corresponding to the two halves of the HIV-1 protease monomer polypeptide chain were joined cleanly and in high yield through unique mutually reactive functional groups, one on each segment. Ligation was performed in 6 molar guanidine hydrochloride, thus circumventing limited solubility of protected peptide segments, the principal problem of the classical approach to the chemical synthesis of proteins. The resulting fully active HIV-1 protease analog contained a thioester replacement for the natural peptide bond between Gly51-Gly52 in each of the two active site flaps, a region known to be highly sensitive to mutational changes of amino acid side chains.

Insights from atomic-resolution X-ray structures of chemically synthesized HIV-1 protease in complex with inhibitors.

The human immunodeficiency virus 1 (HIV-1) protease (PR) is an aspartyl protease essential for HIV-1 viral infectivity. HIV-1 PR has one catalytic site formed by the homodimeric enzyme. We chemically synthesized fully active HIV-1 PR using modern ligation methods. When complexed with the classic substrate-derived inhibitors JG-365 and MVT-101, the synthetic HIV-1 PR formed crystals that diffracted to 1.04- and 1.2-A resolution, respectively. These atomic-resolution structures revealed additional structural details of the HIV-1 PR's interactions with its active site ligands. Heptapeptide inhibitor JG-365, which has a hydroxyethylamine moiety in place of the scissile bond, binds in two equivalent antiparallel orientations within the catalytic groove, whereas the reduced isostere hexapeptide MVT-101 binds in a single orientation. When JG-365 was converted into the natural peptide substrate for molecular dynamic simulations, we found putative catalytically competent reactant states for both lytic water and direct nucleophilic attack mechanisms. Moreover, free energy perturbation calculations indicated that the insertion of catalytic water into the catalytic site is an energetically favorable process.

Molecular tongs containing amino acid mimetic fragments: new inhibitors of wild-type and mutated HIV-1 protease dimerization.

We have designed, synthesized, and evaluated the inhibitory activity and metabolic stability of new peptidomimetic molecular tongs based on a naphthalene scaffold for inhibiting HIV-1 protease dimerization. Peptidomimetic motifs were inserted into one peptidic strand to make it resistant to proteolysis. The peptidic character of the molecular tongs can be decreased without changing the way they inhibit dimerization. Mutated HIV-1 proteases are also vulnerable to dimerization inhibitors, and the multimutated protease ANAM-11 is twice as sensitive to the inhibitor compared to wild-type protease. Thus, the metabolic stability of antidimeric molecular tongs can be increased without compromising their ability to inhibit wild-type and mutated HIV-1 proteases in vitro.

Combating susceptibility to drug resistance: lessons from HIV-1 protease.

Drug resistance is a major obstacle in modern medicine. However, resistance is rarely considered in drug development and may inadvertently be facilitated, as many designed inhibitors contact residues that can mutate to confer resistance, without significantly impairing function. Contemporary drug design often ignores the detailed atomic basis for function and primarily focuses on disrupting the target's activity, which is necessary but not sufficient for developing a robust drug. In this study, we examine the impact of drug-resistant mutations in HIV-1 protease on substrate recognition and demonstrate that most primary active site mutations do not extensively contact substrates, but are critical to inhibitor binding. We propose a general, structure-based strategy to reduce the probability of drug resistance by designing inhibitors that interact only with those residues that are essential for function.

Modular total chemical synthesis of a human immunodeficiency virus type 1 protease.

As part of our ongoing studies of the human immunodeficiency virus type 1 (HIV-1) protease enzyme, we set out to develop a modular chemical synthesis of the protein from multiple peptide segments. Our initial attempts were frustrated by the insolubility of intermediate peptide products. To overcome this problem, we designed a synthetic strategy combining the solubility-enhancing properties of C-terminal (Arg)n tags and the biological phenomenon of autoprocessing of the Gag-Pol polyprotein that occurs during maturation of the HIV-1 virus in vivo. Synthesis of a 119-residue peptide chain containing 10 residues of the reverse transcriptase (RT) open reading frame plus an (Arg)(10) tag at the C-terminus was straightforward by native chemical ligation followed by conversion of the Cys residues to Ala by Raney nickel desulfurization. The product polypeptide itself completed the final synthetic step by removing the C-terminal modification under folding conditions, to give the mature 99-residue polypeptide. High-purity homodimeric HIV-1 protease protein was obtained in excellent yield and had full enzymatic activity; the structure of the synthetic enzyme was confirmed by X-ray crystallography to a resolution of 1.07 A. This efficient modular synthesis by a biomimetic autoprocessing strategy will enable the facile synthesis of unique chemical analogues of the HIV-1 protease to further elucidate the molecular basis of enzyme catalysis.

Monitoring of solid phase peptide synthesis by FT-IR spectroscopy.

Aggregation phenomena of growing peptides on the resin have seldom been investigated. We report here how conformations are determined by FT-IR spectroscopy. Therefore the sequence 80-99 of HIV 1-protease was synthesized. After every coupling a resin sample was taken out of the reaction column and a FT-IR spectrum recorded. The results were compared with the UV monitoring obtained from another synthesis of the same peptide.

Inhibition of HIV-1 proteinase by non-peptide carboxylates.

Some simple dicarboxylates are among the first reported non-peptide inhibitors of HIV-1 proteinase. Only weak inhibition (IC50 greater than or equal to 10 microM) was observed but this may be significant since only two potential enzyme-binding groups are present. Dixon plots and preliminary kinetic data are reported and a possible mechanism for the inhibition is discussed. The dicarboxylates are long enough to engage the carboxylate side chains of Arg 8 and Arg 108 at either end of the 24A long substrate-binding groove. This mode of binding has not been proven but other molecules with similarly separated charged ends are equally effective inhibitors, perhaps indicating a common mechanism of inhibition. There is evidence that placing other functional groups on the inhibitor enables alternative interactions with the enzyme which can reduce inhibitor potency. We propose that incorporation of ionic binding groups in more elaborate and selective non-peptides may potentiate inhibition of HIV-1 proteinase.

In situ neutralization in Boc-chemistry solid phase peptide synthesis. Rapid, high yield assembly of difficult sequences.

Simple, effective protocols have been developed for manual and machine-assisted Boc-chemistry solid phase peptide synthesis on polystyrene resins. These use in situ neutralization [i.e. neutralization simultaneous with coupling], high concentrations (> 0.2 M) of Boc-amino acid-OBt esters plus base for rapid coupling, 100% TFA for rapid Boc group removal, and a single short (30 s) DMF flow wash between deprotection/coupling and between coupling/deprotection. Single 10 min coupling times were used throughout. Overall cycle times were 15 min for manual and 19 min for machine-assisted synthesis (75 residues per day). No racemization was detected in the base-catalyzed coupling step. Several side reactions were studied, and eliminated. These included: pyrrolidonecarboxylic acid formation from Gln in hot TFA-DMF; chain-termination by reaction with excess HBTU; and, chain termination by acetylation (from HOAc in commercial Boc-amino acids). The in situ neutralization protocols gave a significant increase in the efficiency of chain assembly, especially for "difficult" sequences arising from sequence-dependent peptide chain aggregation in standard (neutralization prior to coupling) Boc-chemistry SPPS protocols or in Fmoc-chemistry SPPS. Reported syntheses include HIV-1 protease(1-50,Cys.amide), HIV-1 protease(53-99), and the full length HIV-1 protease(1-99).

Catalytic contribution of flap-substrate hydrogen bonds in "HIV-1 protease" explored by chemical synthesis.

An analogue of "HIV-1 protease" was designed in which the ability to donate important water-mediated hydrogen bonds to substrate was precisely and directly deleted. Chemical ligation of unprotected peptide segments was used to synthesize this "backbone-engineered" enzyme. The functionally relevant amide -CONH- linkage between residues Gly49-Ile50 in each flap of the enzyme was replaced by an isosteric thioester -COS- bond. The backbone-engineered enzyme had normal substrate specificity and affinity (Km). However, the catalytic activity (kcat) was reduced approximately 3000-fold compared to the native amide bond-containing enzyme. Inhibition by the reduced peptide bond substrate analogue MVT-101 was unaffected compared with native enzyme. By contrast, the normally tight-binding hydroxyethylamine inhibitor JG-365 bound to the backbone-engineered enzyme with an approximately 2500-fold reduction in affinity. The reduced catalytic activity of the -Gly49-psi(COS)-Ile50-backbone-engineered enzyme analogue provides direct experimental evidence to support the suggestion that backbone hydrogen bonds from the enzyme flaps to the substrate are important for the catalytic function of the HIV-1 protease.

Total chemical synthesis of enzymes.

The total synthesis, at will, of a wide variety of protein and enzyme molecules is made feasible by modem chemical ligation methods. As Emil Fischer intuitively understood, synthetic access to the enzyme molecule enables the power of chemical science to be applied to elucidating the molecular basis of catalytic function in unprecedented detail.

Analysis of the structure of chemically synthesized HIV-1 protease complexed with a hexapeptide inhibitor. Part I: Crystallographic refinement of 2 A data.

The structure of a complex between a hexapeptide-based inhibitor, MVT-101, and the chemically synthesized (Aba 67,95,167,195; Aba: L-alpha-amino-n-butyric acid) protease from the human immunodeficiency virus (HIV-1), reported previously at 2.3 A has now been refined to a crystallographic R factor of 15.4% at 2.0 A resolution. Root mean square deviations from ideality are 0.18 A for bond lengths and 2.4 degrees for the angles. The inhibitor can be fitted to the difference electron density map in two alternative orientations. Drastic differences are observed for positions and interactions at P3/S3 and P3'/S3' subsites of the two orientations due to different crystallographic environments.