Dual Noncanonical Amino Acid Incorporation Enabling Chemoselective Protein Modification at Two Distinct Sites in Yeast.

Incorporation of more than one noncanonical amino acid (ncAA) within a single protein endows the resulting construct with multiple useful features such as augmented molecular recognition or covalent cross-linking capabilities. Herein, for the first time, we demonstrate the incorporation of two chemically distinct ncAAs into proteins biosynthesized in Saccharomyces cerevisiae. To complement ncAA incorporation in response to the amber (TAG) stop codon in yeast, we evaluated opal (TGA) stop codon suppression using three distinct orthogonal translation systems. We observed selective TGA readthrough without detectable cross-reactivity from host translation components. Readthrough efficiency at TGA was modulated by factors including the local nucleotide environment, gene deletions related to the translation process, and the identity of the suppressor tRNA. These observations facilitated systematic investigation of dual ncAA incorporation in both intracellular and yeast-displayed protein constructs, where we observed efficiencies up to 6% of wild-type protein controls. The successful display of doubly substituted proteins enabled the exploration of two critical applications on the yeast surface─(A) antigen binding functionality and (B) chemoselective modification with two distinct chemical probes through sequential application of two bioorthogonal click chemistry reactions. Lastly, by utilizing a soluble form of a doubly substituted construct, we validated the dual incorporation system using mass spectrometry and demonstrated the feasibility of conducting selective labeling of the two ncAAs sequentially using a "single-pot" approach. Overall, our work facilitates the addition of a 22nd amino acid to the genetic code of yeast and expands the scope of applications of ncAAs for basic biological research and drug discovery.

Exploration of Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase Activity in Yeast.

Archaeal pyrrolysyl-tRNA synthetases (PylRSs) have been used to genetically encode over 200 distinct noncanonical amino acids (ncAAs) in proteins in Escherichia coli and mammalian cells. This vastly expands the range of chemical functionality accessible within proteins produced in these organisms. Despite these clear successes, explorations of PylRS function in yeast remain limited. In this work, we demonstrate that the Methanomethylophilus alvus PylRS (MaPylRS) and its cognate tRNACUAMaPyl support the incorporation of ncAAs into proteins produced in Saccharomyces cerevisiae using stop codon suppression methodologies. Additionally, we prepared three MaPylRS mutants originally engineered in E. coli and determined that all three were active with one or more ncAAs, although with low efficiencies of ncAA incorporation in comparison to the parent MaPylRS. Alongside MaPylRS variants, we evaluated the activity of previously reported Methanosarcina mazei, Methanosarcina barkeri, and chimeric M. mazei and M. barkeri PylRSs. Using S. cerevisiae RJY100 and pairing these PylRSs with the M. mazei tRNACUA, we did not observe any detectable stop codon suppression activity under the same conditions that produced moderately efficient ncAA incorporation with MaPylRS. The addition of MaPylRS/tRNACUAMaPyl to the orthogonal translation machinery toolkit in S. cerevisiae potentially opens the door to hundreds of ncAAs that have not previously been genetically encodable using other aminoacyl-tRNA synthetase/tRNA pairs. Extending the scope of ncAA incorporation in yeast could powerfully advance chemical and biological research for applications ranging from basic biological discovery to enzyme engineering and therapeutic protein lead discovery.

Protein stabilization by tuning the steric restraint at the reverse turn.

Reverse turns are solvent-exposed motifs in proteins that are crucial in nucleating ß-sheets and drive the protein folding. The solvent-exposed nature makes reverse turns more amenable to chemical modifications than α-helices or ß-sheets towards modulating the stability of re-engineered proteins. Here, we utilize van der Waals repulsive forces in tuning the steric restraint at the reverse turn. The steric restraint induced upon N-methylation of the i+1-i+2 amide bond at the reverse turn results in well-folded and stable ß-sheets in aqueous solution at room temperature. The developed superactive turn inducing motif is tolerant to a wide variety of functional groups present on coded amino acids making the designed turn fully compatible with bioactive loops in proteins. We demonstrate that the steric restraint and the functional groups at the reverse turn act in synergy to modulate the folding of re-engineered ß-sheets. Introduction of the turn motifs onto a three-stranded ß-sheet protein, Pin 1 WW domain, resulted in various analogs showing a cooperative two-state transition with thermal stability (TM) ranging from 62 °C to 82 °C. Despite modulating the stability of Pin 1 variants by ∼2.8 kcal mol-1 (ΔΔGf), the native fold in all the protein variants was found to be unperturbed. This structural stability is brought about by conformational preorganization at the engineered reverse turn that results in strong intramolecular hydrogen bonds along the three dimensional structure of the protein. Thus, this simple loop engineering strategy via two amino acid substitution provides us a "toolkit" to modulate the stability of ß-sheet containing peptides and proteins in aqueous solution that will greatly expand the scope of de novo protein and foldamer design.

Dispersive Optical Activity of (R)-Methylene Norbornene: Intrinsic Response and Solvation Effects.

The dispersive optical activity of a homoconjugated bicyclic diene, (R)-methylene norbornene (R-MNB), was interrogated under complementary vapor-phase and solution-phase conditions to elucidate the structural/electronic provenance of its unusual chiroptical signatures and to explore the marked influence of environmental perturbations. The intrinsic (isolated-molecule) values of specific rotation measured at 355 and 633 nm (1623.5 ± 5.5 and 390.4 ± 3.7 deg dm-1 (g/mL)-1) were found to be factors of 3.9 and 2.1 smaller in magnitude than analogous quantities obtained for the kindred enone, (R)-norbornenone (R-NBO), reflecting, in part, the loss of prominent magnetic-dipole contributions from the C═O moiety and the exclusion of electron delocalization from the oxygen lone pairs. The wavelength-resolved rotatory powers of R-MNB were enhanced dramatically (by ∼40% on average) upon dissolution in any of the four common solvents targeted by the present study (acetonitrile, di-n-butylether, cyclohexane, and chloroform), yet displayed only a slight dependence on the exact nature of the surrounding liquid (±2.7% variation from the mean at 589.3 nm). Quantum-chemical calculations built upon the linear-response frameworks of density-functional theory and coupled-cluster theory were enlisted to interpret experimental results, with the substantial effects incurred by nonspecific solvation phenomena being explored through use of polarizable continuum models and bulk property-response relationships. Aside from enumerating the varied quality of agreement attained between computational predictions and polarimetric measurements, these efforts have found that refractive-index correlations, akin to those embodied in the venerable (albeit often discounted) Lorentz local-field correction, afford a viable means of linking the chiroptical behavior of R-MNB across phases.

Immune responses against hepatitis C virus genotype 3a virus-like particles in mice: A novel VLP prime-adenovirus boost strategy.

Chronic hepatitis C virus (HCV) infection represents a major health threat to global population. In India, approximately 15-20% of cases of chronic liver diseases are caused by HCV infection. Although, new drug treatments hold great promise for HCV eradication in infected individuals, the treatments are highly expensive. A vaccine for preventing or treating HCV infection would be of great value, particularly in developing countries. Several preclinical trials of virus-like particle (VLP) based vaccine strategies are in progress throughout the world. Previously, using baculovirus based system, we have reported the production of hepatitis C virus-like particles (HCV-LPs) encoding structural proteins for genotype 3a, which is prevalent in India. In the present study, we have generated HCV-LPs using adenovirus based system and tried different immunization strategies by using combinations of both kinds of HCV-LPs with other genotype 3a-based immunogens. HCV-LPs and peptides based ELISAs were used to evaluate antibody responses generated by these combinations. Cell-mediated immune responses were measured by using T-cell proliferation assay and intracellular cytokine staining. We observed that administration of recombinant adenoviruses expressing HCV structural proteins as final booster enhances both antibody as well as T-cell responses. Additionally, reduction of binding of VLP and JFH1 virus to human hepatocellular carcinoma cells demonstrated the presence of neutralizing antibodies in immunized sera. Taken together, our results suggest that the combined regimen of VLP followed by recombinant adenovirus could more effectively inhibit HCV infection, endorsing the novel vaccine strategy.

Engineering ß-sheets employing N-methylated heterochiral amino acids.

There is a lack of functional group diversity in the reverse turn motifs nucleating a ß-sheet conformation in designed peptides, proteins and foldamers. The majority of these sequences consist of d-Pro-l-Pro, d-Pro-Gly or Asn-Gly as the turn inducing motif restricting their biological application and physicochemical modulation. In this report, for the first time we elucidate that N-methylation of heterochiral amino acids in linear peptides nucleates ß-sheet conformation without the necessity of having a ring or covalent constraint at the reverse turn. Our results show that d-Pro can be conveniently substituted by any other N-methylated d-amino acid followed by an N-methylated l-amino acid or sarcosine to adopt a ßII' turn inducing the ß-sheet folding. Furthermore, we reveal that a single amino acid either at the i + 1 or i + 2 site of the reverse turn can modulate the right-handed twist, which eventually dictates the extent of the foldedness of the ß-hairpin.

Intrinsic Optical Activity and Large-Amplitude Displacement: Conformational Flexibility in (R)-Glycidyl Methyl Ether.

The dispersive optical activity of (R)-(-)-glycidyl methyl ether (R-GME) has been interrogated under ambient vapor-phase and solution-phase conditions, with quantum-chemical analyses built on density functional (B3LYP and CAM-B3LYP) and coupled-cluster (CCSD) implementations of linear-response theory exploited to interpret experimental findings. Inherent flexibility of the heavy atom skeleton leads to nine low-lying structural isomers that possess distinct chiroptical and physicochemical properties, as evinced by marked changes in the magnitude and the sign of rotatory powers observed in various media. These species are interconverted by independent motion along two large-amplitude torsional coordinates and are stabilized differentially by interaction with the surroundings, thereby reapportioning their relative contributions to the collective response evoked from a thermally equilibrated ensemble. The intrinsic behavior exhibited by isolated (vapor-phase) R-GME molecules was calculated through use of both conformer-averaging and restricted vibrational-averaging procedures, the former affording moderately good agreement with measurements of optical rotatory dispersion (ORD) and the latter providing strong evidence for sizable effects arising from vibrational degrees of freedom. A similar conformer-averaging ansatz based on the polarizable-continuum model (PCM) for implicit solvation was deployed to elucidate R-GME specific-rotation parameters acquired for six dilute solutions. This approach gave reasonable predictions for sodium D-line (589.3 nm) experiments performed in the extremes of solvent polarity represented by cyclohexane and acetonitrile but failed to reproduce the overall shape of ORD profiles and suggested more complex processes might be involved in the case of an aromatic medium.

Insights on the origin of the unusually large specific rotation of (1S,4S)-norbornenone.

Measurements and calculations of specific rotation are indispensable for the characterization of chiral molecules and are now performed routinely. However, the factors that determine the magnitude of this property are still not well-understood. The anomalously large specific rotation of (1S,4S)-norbornenone, an outstanding puzzle for over three decades, offers the chance to examine these factors in detail. The present work provides an explanation for the unusual behavior of this molecule in terms of interactions between chemical groups and electronic excited-state transition properties by means of ab initio density functional theory and coupled cluster theory calculations. We show that one can focus on the first excited state and examine the relative orientation of its electric and magnetic transition dipole moments. The contribution of the two transition moments of this electronic state to the specific rotation in a sum-over-states formalism reveals a constructive interaction that is possible only when the two chromophores in norbornenone (C═O and C═C) are in-plane and pointing away from each other. This is due to a small but non-negligible charge transfer between the chromophores and is consistent with recent results from Autschbach's group [Moore et al., J. Chem. Theory Comput. 2012, 8, 4336-4346]. The analysis in this work improves our understanding of this fundamental property of chiral molecules and may help in the design of other molecules with large specific rotation.

Large solvation effect in the optical rotatory dispersion of norbornenone.

The anomalously large chiroptical response of (1R,4R)-norbornenone has been probed under complementary vapor-phase and solution-phase conditions to assess the putative roles of environmental perturbations. Measurements of the specific rotation for isolated (gas-phase) molecules could not be reproduced quantitatively by comprehensive quantum-chemical calculations based on density-functional or coupled-cluster levels of linear-response theory, which suggests that higher-order treatments may be needed to accurately predict such intrinsic behavior. A substantial, yet unexpected, dependence of the dispersive optical activity on the nature (phase) of the surrounding medium has been uncovered, with the venerable Lorentz local-field correction reproducing solvent-mediated trends in rotatory dispersion surprisingly well, whereas more modern polarizable continuum models for implicit solvation performed less satisfactorily.

Intrinsic optical activity and conformational flexibility: the role of size-dependent ring morphology in model cycloketones.

The optical rotatory dispersion of two monocyclic ketones, (R)-3-methylcyclopentanone [R-3MCP] and (R)-3-methylcyclohexanone [R-3MCH], has been investigated under isolated and solvated conditions to explore the role of ring size/morphology and to elucidate the impact of environmental perturbations. Vapor-phase measurements of specific rotation, [α]λT, were performed at 355/633 nm by means of cavity ring-down polarimetry while complementary solution-phase work employed a canonical discrete-wavelength polarimeter to probe five distinct solvents. The magnitude of [α]λT was found to increase upon solvation, albeit to different extents for the two species of interest, with the attendant sign switching between the solution and vapor phases for λ ≥ 510.7 nm in the case of R-3MCH. Quantum-chemical analyses suggest two low-lying conformers to exist for each ketone, distinguished by an equatorial or axial arrangement of the methyl substituent. Linear-response calculations built upon density-functional [DFT(B3LYP)/aug-cc-pVTZ] and coupled-cluster [CCSD/aug-cc-pVDZ] frameworks gave antagonistic chiroptical parameters for these isomers, which were combined with various energy metrics in a conformer-averaging ansatz to simulate the response for a thermally equilibrated ensemble. The intrinsic behavior of R-3MCP was reproduced best by averaging DFT optical-activity predictions according to relative populations deduced from free-energy differences; however, less satisfactory agreement was realized for isolated R-3MCH molecules. The sizable circular birefringence of R-3MCP can be attributed to inherent chirality of its twisted carbon ring whereas the more modest response of R-3MCH stems mainly from the lone stereogenic center. The implicit polarizable continuum model treated solvation effects in R-3MCP with moderate success, but failed to replicate solvent-dependent trends in R-3MCH. The relationship of dispersive optical activity to bulk characteristics of the surrounding medium, including dielectric constant, refractive index, and polarizability, is discussed with the goal of bridging the gap between isolated and solvated chiroptical properties.

A tale of two carenes: intrinsic optical activity and large-amplitude nuclear displacement.

The specific rotation for two isomeric members of the terpene family, (S)-(+)-2-carene and (S)-(+)-3-carene, has been investigated under complementary solvated and isolated conditions, where the latter vapor-phase work has been performed at excitation wavelengths of 355 and 633 nm by means of ultrasensitive cavity ring-down polarimetry (CRDP). Linear-response computations of dispersive optical activity built upon analogous density-functional (B3LYP/aug-cc-pVTZ) and coupled-cluster (CCSD/aug-cc-pVDZ) levels of theory have been enlisted to unravel the structural and electronic origins of observed behavior. The six-membered portion of the bicyclic skeleton in the nominally rigid 3-carene system is predicted to be near-planar in nature, with calculated and measured rotatory powers for the isolated (gas-phase) species shown to be in excellent agreement. In contrast, the inherent flexibility of 2-carene gives rise to two quasidegenerate conformations that are interconnected by a large-amplitude ring-puckering motion and exhibit antagonistic chiroptical properties. Various approaches to simulate the intrinsic response evoked from a thermally equilibrated ensemble of gaseous (S)-(+)-2-carene molecules have been considered, including implicit averaging over independent conformers and explicit (albeit restricted) averaging over nuclear degrees of freedom. A polarizable continuum model for implicit solvation was found to describe solvent-dependent trends reasonably well in the case of (S)-(+)-2-carene, but failed to reproduce the specific-rotation patterns emerging from polarimetric studies of (S)-(+)-3-carene.

Early events associated with the excited state proton transfer in 2-(2'-pyridyl)benzimidazole.

2-(2(')-Pyridyl)benzimidazole (2PBI) undergoes excited state proton transfer (ESPT) in acidic solutions, leading to a tautomer emission at 460 nm. This photoprocess has been studied using ultrafast fluorescence spectroscopic techniques in acidic neat aqueous solutions, in viscous mixtures of glycerol with water, as well as in sucrose solutions. The tautomer is found to be stabilized in the more viscous medium, leading to a greater relative quantum yield as well as lifetime. The long rise time in tautomer emission is not affected by viscosity though. Rather, it appears to have the same value as the long component of the decay of the cationic excited state (C(*)). In addition to the subnanosecond lifetime reported earlier, C(*) is found to exhibit a decay time of 2 ps. This is assigned to its protonation to form the nonfluorescent dication in its excited state (D(*)) considering the ground and excited state pK(a) values reported earlier. An additional rising component of 100 ps is observed in the region of C(*) emission. This is likely to arise from a structural change or charge redistribution in C(*) immediately after its creation and before the phototautomerization.

Chlorin p6 as a fluorescent probe for the investigation of surfactant-cyclodextrin interactions.

Cyclodextrins (CD) are often proposed as potential vehicles in targeted drug delivery. However, if the membrane structure is disrupted by CD, then it cannot be considered to be a good drug delivery vehicle. When an extrinsic fluorescence probe is used to monitor such interactions, there are no less than three possible equilibria that can operate simultaneously: surfactant-cyclodextrin, surfactant-fluorophore and cyclodextrin-fluorophore. The fluorescence intensity/lifetime might be affected by all these and so, the results depend strongly on the fluorophore used as well as the nature of the surfactant. This aspect highlights the importance of the suitability of the fluorescence probe to be used to study complicated systems and interaction. In the present work, chlorin p6, prepared from chlorophyll from spinach leaves, has been used as the fluorescence probe to investigate the interaction between alpha-CD and beta-CD with the neutral surfactants Triton X 100 (TX 100) and cetyl trimethyl ammonium bromide (CTAB). The fluorophore is found to be a sensitive one for the study of the interaction of alpha, beta and gamma-CD with the surfactants TX 100 and CTAB. It is found that contrary to earlier reports, a complex between alpha-CD and TX 100 is formed, even though the binding constant is not very high. This observation can be obtained with chlorin p6, which does not bind to the CDs, but not with a fluorophore, which binds to the CD as well and thus complicates the situation as the binding with CD is stronger than that between TX 100 and alpha-CD as compared to that between TNS and CD.