We used UV resonance Raman (UVRR) spectroscopy to quantitatively correlate the peptide bond AmIII3 frequency to its Psi Ramachandran angle and to the number and types of amide hydrogen bonds at different temperatures. This information allows us to develop a family of relationships to directly estimate the Psi Ramachandran angle from measured UVRR AmIII3 frequencies for peptide bonds (PBs) with known hydrogen bonding (HB). These relationships ignore the more modest Phi Ramachandran angle dependence and allow determination of the Psi angle with a standard error of +/-8 degrees , if the HB state of a PB is known. This is normally the case if a known secondary structure motif is studied. Further, if the HB state of a PB in water is unknown, the extreme alterations in such a state could additionally bias the Psi angle by +/-6 degrees . The resulting ability to measure Psi spectroscopically will enable new incisive protein conformational studies, especially in the field of protein folding. This is because any attempt to understand reaction mechanisms requires elucidation of the relevant reaction coordinate(s). The Psi angle is precisely the reaction coordinate that determines secondary structure changes. As shown elsewhere (Mikhonin et al. J. Am. Chem. Soc. 2005, 127, 7712), this correlation can be used to determine portions of the energy landscape along the Psi reaction coordinate.

UV resonance Raman (UVR) spectroscopy was used to examine the solution conformation of poly-l-lysine (PLL) and poly-l-glutamic acid (PGA) in their non-alpha-helical states. UVR measurements indicate that PLL (at pH = 2) and PGA (at pH = 9) exist mainly in a mixture of polyproline II (PPII) and a novel left-handed 2.5(1)-helical conformation, which is an extended beta-strand-like conformation with Psi approximately +170 degrees and Phi approximately -130 degrees . Both of these conformations are highly exposed to water. The energies of these conformations are very similar. We see no evidence of any disordered "random coil" states. In addition, we find that a PLL and PGA mixture at neutral pH is approximately 60% beta-sheet and contains PPII and extended 2.5(1)-helix conformations. The beta-sheet conformation shows little evidence of amide backbone hydrogen bonding to water. We also developed a method to estimate the distribution of Psi Ramachandran angles for these conformations, which we used to estimate a Psi Ramachandran angle energy landscape. We believe that these are the first experimental studies to give direct information on protein and peptide energy landscapes.

The effect of hydrogen bonding on the amide group vibrational spectra has traditionally been rationalized by invoking a resonance model where hydrogen bonding impacts the amide functional group by stabilizing its [-O-C=NH+] structure over the [O=C-NH] structure. However, Triggs and Valentini’s UV-Raman study of solvation and hydrogen bonding effects on ε-caprolactum, N,N-dimethylacetamide (DMA) and N-methylacetamide (NMA) (J .Phys. Chem., 1992; 96, 6922-31) cast doubt on the validity of this model by demonstrating that contrary to the resonance model prediction, carbonyl hydrogen bonding does not impact the AmII’ frequency of DMA. In this study we utilize density functional theory (DFT) calculations to examine the impact of hydrogen bonding on the C=O and N-H functional groups of NMA, which is typically used as a simple model of the peptide bond. Our calculations indicate that, as expected, the hydrogen bonding frequency dependence of the AmI vibration predominantly derives from the C=O group, whereas the hydrogen bonding frequency dependence of the AmII vibration primarily derives from N-H hydrogen bonding. In contrast, the hydrogen bonding dependence of the conformation sensitive AmIII band derives equally from both C=O and N-H groups, and, thus, is equally responsive to hydrogen bonding at the C=O or N-H site. Our work shows that a clear understanding of the normal mode composition of the amide vibrations is crucial for an accurate interpretation of the hydrogen bonding dependence of amide vibrational frequencies.

3’-Azidothymidine (AZT) was the first approved antiviral for the treatment of human immunodeficiency virus (HIV). Reported efforts in clicking the 3’-azido group of AZT have not yielded 1,2,3-triazoles active against HIV or any other viruses. We report herein the first AZT-derived 1,2,3-triazoles with sub-micromolar potencies against HIV-1. The observed antiviral activities from the cytopathic effect (CPE) based assay were confirmed through a single replication cycle assay. Structure-activity-relationship (SAR) studies revealed two structural features key to antiviral activity: a bulky aromatic ring and the 1,5-substitution pattern on the triazole. Biochemical analysis of the corresponding triphosphates showed lower ATP-mediated nucleotide excision efficiency compared to AZT, which along with molecular modeling, suggests a mechanism of preferred translocation of triazoles into the P-site of HIV reverse transcriptase (RT). This mechanism is corroborated with the observed reduction of fold resistance of the triazole analogue to an AZT-resistant HIV variant (9-fold compared to 56-fold with AZT).

Neutral trialanine (Ala3), which is geometrically constrained to have its peptide bond at Phi and Psi angles of alpha-helix and PPII-like conformers, are studied at the B3LYP/6-31+G(d,p) level of theory to examine vibrational interactions between adjacent peptide units. Delocalization of the amide I, amide II, and amide III3 vibrations are analyzed by calculating their potential energy distributions (PED). The vibrational coupling strengths are estimated from the frequency shifts between the amide vibrations of Ala3 and the local amide bond vibrations of isotopically substituted Ala3 derivatives. Our calculations show the absence of vibrational coupling of the amide I and amide II bands in the PPII conformations. In contrast, the alpha-helical conformation shows strong coupling between the amide I vibrations due to the favorable orientation of the C=O bonds and the strong transitional dipole coupling. The amide III3 vibration shows weak coupling in both the alpha-helix and PPII conformations; this band can be treated as a local independent vibration. Our calculated results in general agree with our previous experimental UV Raman studies of a 21-residue mainly alanine-based peptide (AP).

HIV-1 reverse transcriptase (RT) has been an attractive target for the development of antiretroviral agents. Although this enzyme is bi-functional, having both DNA polymerase and ribonuclease H (RNH) activities, there is no clinically approved inhibitor of the RNH activity. Here, we characterize the structural basis and molecular interaction of an allosteric site inhibitor, BHMP07, with the wild type (WT) RNH fragment. Solution NMR experiments for inhibitor titration on WT RNH showed relatively wide chemical shift perturbations, suggesting a long-range conformational effect on the inhibitor interaction. Comparisons of the inhibitor-induced NMR chemical-shift changes of RNH with those of RNH dimer, in the presence and absence of Mg2+, were performed to determine and verify the interaction site. The NMR results, with assistance of molecular docking, indicate that BHMP07 preferentially binds to a site that is located between the RNH active site and the region encompassing helices B and D (the “substrate-handle region”). The interaction site is consistent with the previous proposed site, identified using a chimeric RNH (p15-EC) [Gong, el (2011) Chem. Biol. Drug Des. 77, 39-47], but with slight differences that reflect the characteristics of the amino acid sequences in p15-EC compared to the WT RNH.

We investigated the normal mode composition and the aqueous solvation dependence of the primary amide vibrations of propanamide. Infrared, normal Raman, and UV resonance Raman (UVRR) spectroscopy were applied in conjunction with density functional theory (DFT) to assign the vibrations of crystalline propanamide. We examined the aqueous solvation dependence of the primary amide UVRR bands by measuring spectra in different acetonitrile/water mixtures. As previously observed in the UVRR spectra of N-methylacetamide, all of the resonance enhanced primary amide bands, except for the Amide I (AmI), show increased UVRR cross sections as the solvent becomes water-rich. These spectral trends are rationalized by a model wherein the hydrogen bonding and the high dielectric constant of water stabilizes the ground state dipolar − O -C=NH 2 + resonance structure over the neutral O=C-NH 2 resonance structure. Thus, vibrations with large C-N stretching show increased UVRR cross sections because the C-N displacement between the electronic ground and excited state increases along the C-N bond. In contrast, vibrations dominated by C=O stretching, such as the AmI, show a decreased displacement between the electronic ground and excited state, which result in a decreased UVRR cross section upon aqueous solvation. The UVRR primary amide vibrations can be used as sensitive spectroscopic markers to study the local dielectric constant and hydrogen bonding environments of the primary amide side chains of glutamine (Gln) and asparagine (Asn). Graphical Abstract *Corresponding Author: asher@pitt.edu. NotesThe authors declare no competing financial interest. Tables S1-S6 and Figures S1-S3. Description of X-ray crystallographic methods to determine structure of propanamide and UVRR spectral deconvolution methods. This material is available free of charge via the Internet at http://pubs.acs.org/. Supporting Information

We utilized UV-resonance Raman (UVRR) measurements and density functional theory (DFT) calculations to relate that the AmII′p frequency to the ψ-angle. The AmII′p frequency shifts by ∼ 25 cm -1 as the ψ-angle is varied over allowed angles of the pro peptide bond. The AmII′p frequency does not show any significant dependence on the φ-dihedral angle. The conformation sensitivity of the AmII′p frequency derives from conformation-induced changes in the planarity of the Pro peptide bond; ψ angles changes push the amide nitrogen out of the peptide bond plane. We use this AmII′p frequency dependence on the ψ-angle to track temperature-induced conformation changes in a polyproline peptide. The temperature-induced 7 cm -1 downshift in the AmII′p frequency of the polyproline peptide results from a ∼45° rotation of the ψ dihedral angle from ψ = 145° (ideal PPII conformation) to ψ = 100° (collapsed PPII conformation).