A Chemical Counterpart to the Resolution Step of Nature's Intein-Mediated Protein Splicing.

In the course of an attempted total chemical synthesis of the ant insulin-like peptide-2 (ILP2) protein molecule, specific cleavage of a backbone peptide bond in a branched ester-linked polypeptide chain with concomitant peptide splicing was observed. The side reaction was investigated in model compounds. Here, we postulate a chemical mechanism for this novel polypeptide backbone cleavage reaction as a chemical counterpart to the resolution step of biochemical intein-mediated protein splicing.

A Non-immunogenic Bivalent d-Protein Potently Inhibits Retinal Vascularization and Tumor Growth.

We report a general approach to engineering multivalent d-proteins with antibody-like activities in vivo. Mirror-image phage display and structure-guided design were utilized to create a d-protein that uses receptor mimicry to antagonize vascular endothelial growth factor A (VEGF-A). Selections against the d-protein form of VEGF-A using phage-displayed libraries of two different domain scaffolds yielded two proteins that bound distinct receptor interaction sites on VEGF-A. X-ray crystal structures of the d-protein/VEGF-A complexes were used to guide affinity maturation and to construct a heterodimeric d-protein VEGF-A antagonist with picomolar activity. The d-protein VEGF-A antagonist prevented vascular leakage in a rabbit eye model of wet age-related macular degeneration and slowed tumor growth in the MC38 syngeneic mouse tumor model with efficacies comparable to those of approved antibody drugs, and in contrast with antibodies, the d-protein was non-immunogenic during treatment and following subcutaneous immunizations.

Visualizing Tetrahedral Oxyanion Bound in HIV-1 Protease Using Neutrons: Implications for the Catalytic Mechanism and Drug Design.

HIV-1 protease is indispensable for virus propagation and an important therapeutic target for antiviral inhibitors to treat AIDS. As such inhibitors are transition-state mimics, a detailed understanding of the enzyme mechanism is crucial for the development of better anti-HIV drugs. Here, we used room-temperature joint X-ray/neutron crystallography to directly visualize hydrogen atoms and map hydrogen bonding interactions in a protease complex with peptidomimetic inhibitor KVS-1 containing a reactive nonhydrolyzable ketomethylene isostere, which, upon reacting with the catalytic water molecule, is converted into a tetrahedral intermediate state, KVS-1TI. We unambiguously determined that the resulting tetrahedral intermediate is an oxyanion, rather than the gem-diol, and both catalytic aspartic acid residues are protonated. The oxyanion tetrahedral intermediate appears to be unstable, even though the negative charge on the oxyanion is delocalized through a strong n → π* hyperconjugative interaction into the nearby peptidic carbonyl group of the inhibitor. To better understand the influence of the ketomethylene isostere as a protease inhibitor, we have also examined the protease structure and binding affinity with keto-darunavir (keto-DRV), which similar to KVS-1 includes the ketomethylene isostere. We show that keto-DRV is a significantly less potent protease inhibitor than DRV. These findings shed light on the reaction mechanism of peptide hydrolysis catalyzed by HIV-1 protease and provide valuable insights into further improvements in the design of protease inhibitors.

Novel protein science enabled by total chemical synthesis.

Chemical synthesis is a well-established method for the preparation in the research laboratory of multiple-tens-of-milligram amounts of correctly folded, high purity protein molecules. Chemically synthesized proteins enable a broad spectrum of novel protein science. Racemic mixtures consisting of d-protein and l-protein enantiomers facilitate crystallization and determination of protein structures by X-ray diffraction. d-Proteins enable the systematic development of unnatural mirror image protein molecules that bind with high affinity to natural protein targets. The d-protein form of a therapeutic target can also be used to screen natural product libraries to identify novel small molecule leads for drug development. Proteins with novel polypeptide chain topologies including branched, circular, linear-loop, and interpenetrating polypeptide chains can be constructed by chemical synthesis. Medicinal chemistry can be applied to optimize the properties of therapeutic protein molecules. Chemical synthesis has been used to redesign glycoproteins and for the a priori design and construction of covalently constrained novel protein scaffolds not found in nature. Versatile and precise labeling of protein molecules by chemical synthesis facilitates effective application of advanced physical methods including multidimensional nuclear magnetic resonance and time-resolved FTIR for the elucidation of protein structure-activity relationships. The chemistries used for total synthesis of proteins have been adapted to making artificial molecular devices and protein-inspired nanomolecular constructs. Research to develop mirror image life in the laboratory is in its very earliest stages, based on the total chemical synthesis of d-protein forms of polymerase enzymes.

Reinvestigation of the biological activity of d-allo-ShK protein.

ShK toxin from the sea anemone Stichodactyla helianthus is a 35-residue protein that binds to the Kv1.3 ion channel with high affinity. Recently we determined the X-ray structure of ShK toxin by racemic crystallography, in the course of which we discovered that d-ShK has a near-background IC50 value ∼50,000 times lower than that of the l-ShK toxin. This lack of activity was at odds with previously reported results for an ShK diastereomer designated d-allo-ShK, for which significant biological activity had been observed in a similar receptor-blocking assay. As reported, d-allo-ShK was made up of d-amino acids, but with retention of the natural stereochemistry of the chiral side chains of the Ile and Thr residues, i.e. containing d-allo-Ile and d-allo-Thr along with d-amino acids and glycine. To understand its apparent biological activity, we set out to chemically synthesize d-allo-ShK and determine its X-ray structure by racemic crystallography. Using validated allo-Thr and allo-Ile, both l-allo-ShK and d-allo-ShK polypeptide chains were prepared by total chemical synthesis. Neither the l-allo-ShK nor the d-allo-ShK polypeptides folded, whereas both l-ShK and d-ShK folded smoothly under the same conditions. Re-examination of NMR spectra of the previously reported d-allo-ShK protein revealed that diagnostic Thr and Ile signals were the same as for authentic d-ShK. On the basis of these results, we conclude that the previously reported d-allo-ShK was in fact d-ShK, the true enantiomer of natural l-ShK toxin, and that the apparent biological activity may have arisen from inadvertent contamination with trace amounts of l-ShK toxin.

Perplexing cooperative folding and stability of a low-sequence complexity, polyproline 2 protein lacking a hydrophobic core.

The burial of hydrophobic side chains in a protein core generally is thought to be the major ingredient for stable, cooperative folding. Here, we show that, for the snow flea antifreeze protein (sfAFP), stability and cooperativity can occur without a hydrophobic core, and without α-helices or ß-sheets. sfAFP has low sequence complexity with 46% glycine and an interior filled only with backbone H-bonds between six polyproline 2 (PP2) helices. However, the protein folds in a kinetically two-state manner and is moderately stable at room temperature. We believe that a major part of the stability arises from the unusual match between residue-level PP2 dihedral angle bias in the unfolded state and PP2 helical structure in the native state. Additional stabilizing factors that compensate for the dearth of hydrophobic burial include shorter and stronger H-bonds, and increased entropy in the folded state. These results extend our understanding of the origins of cooperativity and stability in protein folding, including the balance between solvent and polypeptide chain entropies.

Inversion of the Side-Chain Stereochemistry of Indvidual Thr or Ile Residues in a Protein Molecule: Impact on the Folding, Stability, and Structure of the ShK Toxin.

ShK toxin is a cysteine-rich 35-residue protein ion-channel ligand isolated from the sea anemone Stichodactyla helianthus. In this work, we studied the effect of inverting the side chain stereochemistry of individual Thr or Ile residues on the properties of the ShK protein. Molecular dynamics simulations were used to calculate the free energy cost of inverting the side-chain stereochemistry of individual Thr or Ile residues. Guided by the computational results, we used chemical protein synthesis to prepare three ShK polypeptide chain analogues, each containing either an allo-Thr or an allo-Ile residue. The three allo-Thr or allo-Ile-containing ShK polypeptides were able to fold into defined protein products, but with different folding propensities. Their relative thermal stabilities were measured and were consistent with the MD simulation data. Structures of the three ShK analogue proteins were determined by quasi-racemic X-ray crystallography and were similar to wild-type ShK. All three ShK analogues retained ion-channel blocking activity.

Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET.

Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: ß-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.

Scope and Limitations of Fmoc Chemistry SPPS-Based Approaches to the Total Synthesis of Insulin Lispro via Ester Insulin.

We have systematically explored three approaches based on 9-fluorenylmethoxycarbonyl (Fmoc) chemistry solid phase peptide synthesis (SPPS) for the total chemical synthesis of the key depsipeptide intermediate for the efficient total chemical synthesis of insulin. The approaches used were: stepwise Fmoc chemistry SPPS; the "hybrid method", in which maximally protected peptide segments made by Fmoc chemistry SPPS are condensed in solution; and, native chemical ligation using peptide-thioester segments generated by Fmoc chemistry SPPS. A key building block in all three approaches was a Glu[O-ß-(Thr)] ester-linked dipeptide equipped with a set of orthogonal protecting groups compatible with Fmoc chemistry SPPS. The most effective method for the preparation of the 51 residue ester-linked polypeptide chain of ester insulin was the use of unprotected peptide-thioester segments, prepared from peptide-hydrazides synthesized by Fmoc chemistry SPPS, and condensed by native chemical ligation. High-resolution X-ray crystallography confirmed the disulfide pairings and three-dimensional structure of synthetic insulin lispro prepared from ester insulin lispro by this route. Further optimization of these pilot studies could yield an efficient total chemical synthesis of insulin lispro (Humalog) based on peptide synthesis by Fmoc chemistry SPPS.

Obviation of hydrogen fluoride in Boc chemistry solid phase peptide synthesis of peptide-αthioesters.

Under suitable conditions, trifluoromethanesulfonic acid performs comparably to hydrogen fluoride for the on-resin global deprotection of peptides prepared by Boc chemistry solid phase peptide synthesis (SPPS). Obviation of hydrogen fluoride in Boc chemistry SPPS enables the straightforward synthesis of peptide-αthioesters for use in native chemical ligation.

Chemical synthesis and enzymatic properties of RNase A analogues designed to enhance second-step catalytic activity.

In this paper, we have used total chemical synthesis of RNase A analogues in order to probe the molecular basis of enzyme catalysis. Our goal was to obligately fill the adenine-binding pocket on the enzyme molecule, and to thus pre-orient the imidazole side chain of His119 in its catalytically productive orientation. Two designed analogues of the RNase A protein molecule that contained an adenine moiety covalently bound to distinct amino acid side chains adjacent to the adenine binding pocket were prepared. A crystal structure of one analogue was determined at 2.3 Å resolution. Kinetic data for RNA transphosporylation and 2',3' cyclic mononucleotide hydrolysis were acquired for the adenine-containing RNase A analogue proteins. As anticipated, the presence of a covalently attached adenine on the enzyme molecule decreased the rate of transphosphorylation and increased the rate of hydrolysis, although the magnitude of the effects was small. This work illustrates the use of total protein synthesis to investigate the chemistry of enzyme catalysis in ways not possible through traditional biochemistry or molecular biology.

Elucidation of the Covalent and Tertiary Structures of Biologically Active Ts3 Toxin.

Ts3 is an alpha scorpion toxin from the venom of the Brazilian scorpion Tityus serrulatus. Ts3 binds to the domain IV voltage sensor of voltage-gated sodium channels (Nav ) and slows down their fast inactivation. The covalent structure of the Ts3 toxin is uncertain, and the structure of the folded protein molecule is unknown. Herein, we report the total chemical synthesis of four candidate Ts3 toxin protein molecules and the results of structure-activity studies that enabled us to establish the covalent structure of biologically active Ts3 toxin. We also report the synthesis of the mirror image form of the Ts3 protein molecule, and the use of racemic protein crystallography to determine the folded (tertiary) structure of biologically active Ts3 toxin by X-ray diffraction.

ß1-subunit-induced structural rearrangements of the Ca2+- and voltage-activated K+ (BK) channel.

Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory ß-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or ß1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) ß1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the ß1-subunit within the α/ß1-subunit complex.

A Potent d-Protein Antagonist of VEGF-A is Nonimmunogenic, Metabolically Stable, and Longer-Circulating in Vivo.

Polypeptides composed entirely of d-amino acids and the achiral amino acid glycine (d-proteins) inherently have in vivo properties that are proposed to be near-optimal for a large molecule therapeutic agent. Specifically, d-proteins are resistant to degradation by proteases and are anticipated to be nonimmunogenic. Furthermore, d-proteins are manufactured chemically and can be engineered to have other desirable properties, such as improved stability, affinity, and pharmacokinetics. Thus, a well-designed d-protein therapeutic would likely have significant advantages over l-protein drugs. Toward the goal of developing d-protein therapeutics, we previously generated RFX001.D, a d-protein antagonist of natural vascular endothelial growth factor A (VEGF-A) that inhibited binding to its receptor. However, RFX001.D is unstable at physiological temperatures (Tm = 33 °C). Here, we describe RFX037.D, a variant of RFX001.D with extreme thermal stability (Tm > 95 °C), high affinity for VEGF-A (Kd = 6 nM), and improved receptor blocking. Comparison of the two enantiomeric forms of RFX037 revealed that the d-protein is more stable in mouse, monkey, and human plasma and has a longer half-life in vivo in mice. Significantly, RFX037.D was nonimmunogenic in mice, whereas the l-enantiomer generated a strong immune response. These results confirm the potential utility of synthetic d-proteins as alternatives to therapeutic antibodies.

Crystallization of Enantiomerically Pure Proteins from Quasi-Racemic Mixtures: Structure Determination by X-Ray Diffraction of Isotope-Labeled Ester Insulin and Human Insulin.

As a part of a program aimed towards the study of the dynamics of human insulin-protein dimer formation using two-dimensional infrared spectroscopy, we used total chemical synthesis to prepare stable isotope labeled [(1-(13) C=(18) O)Phe(B24) )] human insulin, via [(1-(13) C=(18) O)Phe(B24) )] ester insulin as a key intermediate product that facilitates folding of the synthetic protein molecule (see preceding article). Here, we describe the crystal structure of the synthetic isotope-labeled ester insulin intermediate and the product synthetic human insulin. Additionally, we present our observations on hexamer formation with these two proteins in the absence of phenol derivatives and/or Zn metal ions. We also describe and discuss the fractional crystallization of quasi-racemic protein mixtures containing each of these two synthetic proteins.

Efficient Total Chemical Synthesis of (13) C=(18) O Isotopomers of Human Insulin for Isotope-Edited FTIR.

Isotope-edited two-dimensional Fourier transform infrared spectroscopy (2 D FTIR) can potentially provide a unique probe of protein structure and dynamics. However, general methods for the site-specific incorporation of stable (13) C=(18) O labels into the polypeptide backbone of the protein molecule have not yet been established. Here we describe, as a prototype for the incorporation of specific arrays of isotope labels, the total chemical synthesis-via a key ester insulin intermediate-of 97 % enriched [(1-(13) C=(18) O)Phe(B24) ] human insulin: stable-isotope labeled at a single backbone amide carbonyl. The amino acid sequence as well as the positions of the disulfide bonds and the correctly folded structure were unambiguously confirmed by the X-ray crystal structure of the synthetic protein molecule. In vitro assays of the isotope labeled [(1-(13) C=(18) O)Phe(B24) ] human insulin showed that it had full insulin receptor binding activity. Linear and 2 D IR spectra revealed a distinct red-shifted amide I carbonyl band peak at 1595 cm(-1) resulting from the (1-(13) C=(18) O)Phe(B24) backbone label. This work illustrates the utility of chemical synthesis to enable the application of advanced physical methods for the elucidation of the molecular basis of protein function.

Enhanced Solvation of Peptides Attached to "Solid-Phase" Resins: Straightforward Syntheses of the Elastin Sequence Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val.

The solubility-enhancing power of covalent attachment to solvent-swollen cross-linked resin supports was illustrated by syntheses of the highly aggregating elastin-derived 10-residue peptide sequence Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val using standard protocols for both Boc and Fmoc chemistry SPPS.

A functional role of Rv1738 in Mycobacterium tuberculosis persistence suggested by racemic protein crystallography.

Protein 3D structure can be a powerful predictor of function, but it often faces a critical roadblock at the crystallization step. Rv1738, a protein from Mycobacterium tuberculosis that is strongly implicated in the onset of nonreplicating persistence, and thereby latent tuberculosis, resisted extensive attempts at crystallization. Chemical synthesis of the L- and D-enantiomeric forms of Rv1738 enabled facile crystallization of the D/L-racemic mixture. The structure was solved by an ab initio approach that took advantage of the quantized phases characteristic of diffraction by centrosymmetric crystals. The structure, containing L- and D-dimers in a centrosymmetric space group, revealed unexpected homology with bacterial hibernation-promoting factors that bind to ribosomes and suppress translation. This suggests that the functional role of Rv1738 is to contribute to the shutdown of ribosomal protein synthesis during the onset of nonreplicating persistence of M. tuberculosis.

Photosensitivity of neurons enabled by cell-targeted gold nanoparticles.

Unmodified neurons can be directly stimulated with light to produce action potentials, but such techniques have lacked localization of the delivered light energy. Here we show that gold nanoparticles can be conjugated to high-avidity ligands for a variety of cellular targets. Once bound to a neuron, these particles transduce millisecond pulses of light into heat, which changes membrane capacitance, depolarizing the cell and eliciting action potentials. Compared to non-functionalized nanoparticles, ligand-conjugated nanoparticles highly resist convective washout and enable photothermal stimulation with lower delivered energy and resulting temperature increase. Ligands targeting three different membrane proteins were tested; all showed similar activity and washout resistance. This suggests that many types of ligands can be bound to nanoparticles, preserving ligand and nanoparticle function, and that many different cell phenotypes can be targeted by appropriate choice of ligand. The findings have applications as an alternative to optogenetics and potentially for therapies involving neuronal photostimulation.