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Upconverting nanoparticles (UCNPs) are an emerging platform for mechanical force sensing at the nanometer scale. An outstanding challenge in realizing nanometer-scale mechano-sensitive UCNPs is maintaining a high mechanical force responsivity in conjunction with bright optical emission. This Letter reports mechano-sensing UCNPs based on the lanthanide dopants Yb(3+) and Er(3+), which exhibit a strong ratiometric change in emission spectra and bright emission under applied pressure. We synthesize and analyze the pressure response of five different types of nanoparticles, including cubic NaYF(4) host nanoparticles and alkaline-earth host materials CaLuF, SrLuF, SrYbF, and BaLuF, all with lengths of 15 nm or less. By combining optical spectroscopy in a diamond anvil cell with single-particle brightness, we determine the noise equivalent sensitivity (GPa/√Hz) of these particles. The SrYb(0.72)Er(0.28)F@SrLuF particles exhibit an optimum noise equivalent sensitivity of 0.26 ± 0.04 GPa/√Hz. These particles present the possibility of robust nanometer-scale mechano-sensing.

Cytoplasmic dynein is essential for intracellular transport, but because of its complexity, we still do not fully understand how this 1.5 megadalton protein works. Here, we used novel optical probes that enable single-particle tracking (SPT) of individual cargos transported by dynein motors in live neurons over 900μm. Analyses using the Fluctuation Theorem (FT) showed that the number of dynein molecules switches between 1-5 motors during the transport. Clearly resolved single-molecular steps revealed that the dwell times between individual steps were accurately described by an enzymatic cycle dominated by two equal and thermally-activated rate constants. Based on these data, we propose a new molecular model whereby each step requires the hydrolysis of 2 ATPs. The model is consistent with extensive structural, single-molecule and biochemical measurements.

Lanthanide-doped upconversion nanoparticles (UCNPs) are promising single-molecule probes given their non-blinking, photobleaching-resistant luminescence on infrared excitation. However, the weak luminescence of sub-50 nm UCNPs limits their single-particle detection to above 10 kW cm−2, which is impractical for live cell imaging. Here, we systematically characterize single-particle luminescence for UCNPs with various formulations over a 106 variation in incident power, down to 8 W cm−2. A core–shell–shell (CSS) structure (NaYF4@NaYb1−xF4:Erx@NaYF4) is shown to be significantly brighter than the commonly used NaY0.78F4:Yb0.2Er0.02. At 8 W cm−2, the 8% Er3+ CSS particles exhibit a 150-fold enhancement given their high sensitizer Yb3+ content and the presence of an inert shell to prevent energy migration to defects. Moreover, we reveal power-dependent luminescence enhancement from the inert shell, which explains the discrepancy in enhancement factors reported by ensemble and previous single-particle measurements. These brighter probes open the possibility of cellular and single-molecule tracking at low irradiance. This systematic study of upconversion nanoparticles reveals power-dependent luminescence and paves the way towards ideal single-molecule and cellular probes.

We synthesized and characterized a core-shell-shell upconverting nanoparticle architecture. Compared to the traditional core-shell architecture, we demonstrated 2-fold brighter single particle emission and 8-fold improved emission enhancement via Förster resonant energy transfer to a dye.

Upconverting nanoparticles provide valuable benefits as optical probes for bioimaging and Förster resonant energy transfer (FRET) due to their high signal-to-noise ratio, photostability, and biocompatibility; yet, making nanoparticles small yields a significant decay in brightness due to increased surface quenching. Approaches to improve the brightness of UCNPs exist but often require increased nanoparticle size. Here we present a unique core–shell–shell nanoparticle architecture for small (sub-20 nm), bright upconversion with several key features: (1) maximal sensitizer concentration in the core for high near-infrared absorption, (2) efficient energy transfer between core and interior shell for strong emission, and (3) emitter localization near the nanoparticle surface for efficient FRET. This architecture consists of β-NaYbF(4) (core) @NaY(0.8–x)Er(x)Gd(0.2)F(4) (interior shell) @NaY(0.8)Gd(0.2)F(4) (exterior shell), where sensitizer and emitter ions are partitioned into core and interior shell, respectively. Emitter concentration is varied (x = 1, 2, 5, 10, 20, 50, and 80%) to investigate influence on single particle brightness, upconversion quantum yield, decay lifetimes, and FRET coupling. We compare these seven samples with the field-standard core–shell architecture of βNaY(0.58)Gd(0.2)Yb(0.2)Er(0.02)F(4) (core) @NaY(0.8)Gd(0.2)F(4) (shell), with sensitizer and emitter ions codoped in the core. At a single particle level, the core–shell–shell design was up to 2-fold brighter than the standard core–shell design. Further, by coupling a fluorescent dye to the surface of the two different architectures, we demonstrated up to 8-fold improved emission enhancement with the core–shell–shell compared to the core–shell design. We show how, given proper consideration for emitter concentration, we can design a unique nanoparticle architecture to yield comparable or improved brightness and FRET coupling within a small volume.

Lanthanide-doped upconversion nanoparticles (UCNPs) are promising single-molecule probes given their non-blinking, photobleach-resistant luminescence upon infrared excitation. However, the weak luminescence of sub-50 nm UCNPs limits their single-particle detection to above 10 kWcm(−2) that is impractical for live cell imaging. Here, we systematically characterize single-particle luminescence for UCNPs with various formulations over a 10(6) variation in incident power, down to 8 Wcm(−2). A core-shell-shell (CSS) structure (NaYF(4)@NaYb(1-x)F(4):Er(x)@NaYF(4)) is shown to be significantly brighter than the commonly used NaY(0.78)F(4):Yb(0.2)Er(0.02). At 8 Wcm(−2), the 8% Er(3+) CSS particles exhibit a 150-fold enhancement given their high sensitizer Yb(3+) content and the presence of an inert shell to prevent energy migration to defects. Moreover, we reveal power-dependent luminescence enhancement from the inert shell, which explains the discrepancy in enhancement factors reported by ensemble and previous single-particle measurements. These brighter probes open the possibility of cellular and single-molecule tracking at low irradiance.

Antiviral drugs designed to accelerate viral mutation rates can drive a viral population to extinction in a process called lethal mutagenesis. One such molecule is 5,6-dihydro-5-aza-2'-deoxycytidine (KP1212), a selective mutagen that induces A-to-G and G-to-A mutations in the genome of replicating HIV. The mutagenic property of KP1212 was hypothesized to originate from its amino-imino tautomerism, which would explain its ability to base pair with either G or A. To test the multiple tautomer hypothesis, we used 2D IR spectroscopy, which offers subpicosecond time resolution and structural sensitivity to distinguish among rapidly interconverting tautomers. We identified several KP1212 tautomers and found that60% of neutral KP1212 is present in the enol-imino form. The abundant proportion of this traditionally rare tautomer offers a compelling structure-based mechanism for pairing with adenine. Additionally, the pKa of KP1212 was measured to be 7.0, meaning a substantial population of KP1212 is protonated at physiological pH. Furthermore, the mutagenicity of KP1212 was found to increase dramatically at pH7, suggesting a significant biological role for the protonated KP1212 molecules. Overall, our data reveal that the bimodal mutagenic properties of KP1212 result from its unique shape shifting ability that utilizes both tautomerization and protonation.

The p53 tumor suppressor protein is a central regulator that turns on vast gene networks to maintain cellular integrity upon various stimuli. p53 activates transcription initiation in part by aiding recruitment of TFIID to the promoter. However, the precise means by which p53 dynamically interacts with TFIID to facilitate assembly on target gene promoters remains elusive. To address this key question, we have undertaken an integrated approach involving single molecule fluorescence microscopy, single particle cryo-electron microscopy, and biochemistry. Our real-time single molecule imaging demonstrates that TFIID alone binds poorly to native p53 target promoters. p53 unlocks TFIID’s ability to bind DNA by increasing TFIID contacts with both the core promoter and a region surrounding p53’s response element (RE). Analysis of single molecule dissociation kinetics reveals that TFIID interacts with promoters via transient and prolonged DNA binding modes that are each regulated by p53. Importantly, our structural work reveals that TFIID’s conversion from a canonical form to a rearranged DNA-binding conformation is enhanced in the presence of DNA and p53. Notably, TFIID’s interaction with DNA induces p53 to rapidly dissociate, effectively liberating the RE on the promoter. Collectively, these findings indicate that p53 dynamically escorts and loads the basal transcription machinery onto its target promoters.

Insulin homodimer associates through the coupled folding and binding of two partially disordered monomers. We aim to understand this dynamics by observing insulin dimer dissociation initiated with a nanosecond temperature jump using transient two-dimensional infrared spectroscopy (2D IR) of amide I vibrations. With the help of equilibrium FTIR and 2D IR spectra, and through a systematic study of the dependence of dissociation kinetics on temperature and insulin concentration, we are able to decompose and analyze the spectral evolution associated with different secondary structures. We find that the dissociation under all conditions is characterized by two processes whose influence on the kinetics varies with temperature: the unfolding of the β sheet at the dimer interface observed as exponential kinetics between 250 and 1000 μs and nonexponential kinetics between 5 and 150 μs that we attribute to monomer disordering. Microscopic reversibility arguments lead us to conclude that dimer association requires significant conformational changes within the monomer in concert with the folding of the interfacial β sheet. While our data indicates a more complex kinetics, we apply a two-state model to the β-sheet unfolding kinetics to extract thermodynamic parameters and kinetic rate constants. The association rate constant, ka (23 °C) = 8.8 × 10(5) M(-1) s(-1) (pH 0, 20% EtOD), is approximately 3 orders of magnitude slower than the calculated diffusion limited association rate, which is explained by the significant destabilizing effect of ethanol on the dimer state and the highly positive charge of the monomers at this pH.

The tautomerism of aromatic heterocycles is of great interest because it directly affects their chemical properties and biological function. The tautomerism of 2-pyridone, 6-chloro-2-pyridone, and 4-pyrimidinone have been examined in D(2)O using FTIR, two-dimensional IR (2D IR) spectroscopy and density functional theory (DFT) calculations. Using the 2D IR cross-peak patterns, the lactim tautomer of 6-chloro-2-pyridone was separated from the lactam tautomer, and its population was observed to increase with temperature. The equilibrium constant of [lac-tam]/[lactim] was determined to be 2.1 at room temperature for 6-chloro-2-pyridone. Similarly, the N1H and N3H lactam tautomers of 4-pyrimidinone were identified with 2D IR. To assign the vibrational modes of different tautomers, DFT calculations of these chemical species were performed with explicit water molecules, and the hydration effects on the vibrational frequencies and intensities were established.

More than 50% of cancer patients harbor p53 mutations, highlighting the essential role of p53 in tumor suppression. In response to various stress signals, p53 targets the TFIID-mediated transcription machinery to stimulate expression of vast gene networks involved in diverse cellular pathways. Understanding how p53 turns on TFIID-mediated transcription initiation at the single molecule level will advance our knowledge of p53's tumor suppression activity.Despite 20 years of biochemical studies, it remains elusive how p53 recruits TFIID and other basal transcription factors (e.g. TFIIB and RNA Polymerase II) to facilitate pre-initiation complex formation on various target gene promoters. We hypothesize that p53 induces distinct DNA-binding conformations within basal factors to stimulate assembly culminated in transcription initiation. Therefore, to test our hypothesis, we employed an innovative integrated approach involving single particle cryo-electron microscopy (EM), single molecule fluoresce microscopy and biochemistry.Intriguingly, our studies demonstrated that p53 delivers and promotes stable binding of TFIID to DNA. Unexpectedly, the interaction of TFIID and DNA in turn causes p53 to quickly dissociate from the assembly and promoter. Furthermore, our work suggest that the direct association of p53 and TFIID is sufficient to recruit TFIID onto various target gene promoters. More significantly, p53 induces a common DNA-binding conformation of TFIID. We further identified a novel role of TFIID in anchoring core promoter DNA downstream of the transcription start site.Collectively, these findings indicate that p53, and potentially activators in general, serve as escorts to dynamically recruit and load the basal transcription machinery onto DNA. Importantly, p53 facilitates formation of common DNA-binding forms of basal factors. Overall, our studies are critical for understanding how eukaryotic transcription complexes initiate transcription on different types of protein-coding genes in response to various stress signals.

In the active cytosine demethylation pathway, 5-methylcytosine (5mC) is oxidized sequentially to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Thymine DNA glycosylase (TDG) selectively excises 5fC and 5caC but not cytosine (C), 5mC, and 5hmC. We propose that the electron-withdrawing properties of -CHO and -COOH in 5fC and 5caC increase N3 acidity, leading to weakened hydrogen bonding and reduced base pair stability relative to C, 5mC, and 5hmC, thereby facilitating the selective recognition of 5fC and 5caC by TDG. Through (13)C NMR, we measured the pKa at N3 of 5fC as 2.4 and the two pKa's of 5caC as 2.1 and 4.2. We used isotope-edited IR spectroscopy coupled with density functional theory (DFT) calculations to site-specifically assign the more acidic pKa of 5caC to protonation at N3, indicating that N3 acidity is increased in 5fC and 5caC relative to C. IR and UV melting studies of self-complementary DNA oligomers confirm reduced stability for 5fC-G and 5caC-G base pairs. Furthermore, while the 5fC-G base pair stability is insensitive to pH, the 5caC-G stability is reduced as pH decreases and the carboxyl group is increasingly protonated. Despite suggestions that 5fC and 5caC may exist in rare tautomeric structures which form wobble GC base pairs, our two-dimensional infrared (2D IR) spectroscopy of 5fC and 5caC free nucleosides confirms that both bases are predominantly in the canonical amino-keto form. Taken together, these findings support our model that weakened base pairing ability for 5fC and 5caC in dsDNA contributes to their selective recognition by TDG.

Viral lethal mutagenesis is a strategy whereby the innate immune system or mutagenic pool nucleotides increase the error rate of viral replication above the error catastrophe limit. Lethal mutagenesis has been proposed as a mechanism for several antiviral compounds, including the drug candidate 5-aza-5,6-dihydro-2'-deoxycytidine (KP1212), which causes A-to-G and G-to-A mutations in the HIV genome, both in tissue culture and in HIV positive patients undergoing KP1212 monotherapy. This work explored the molecular mechanism(s) underlying the mutagenicity of KP1212, and specifically whether tautomerism, a previously proposed hypothesis, could explain the biological consequences of this nucleoside analog. Establishing tautomerism of nucleic acid bases under physiological conditions has been challenging because of the lack of sensitive methods. This study investigated tautomerism using an array of spectroscopic, theoretical, and chemical biology approaches. Variable temperature NMR and 2D infrared spectroscopic methods demonstrated that KP1212 existed as a broad ensemble of interconverting tautomers, among which enolic forms dominated. The mutagenic properties of KP1212 were determined empirically by in vitro and in vivo replication of a single-stranded vector containing a single KP1212. It was found that KP1212 paired with both A (10%) and G (90%), which is in accord with clinical observations. Moreover, this mutation frequency is sufficient for pushing a viral population over its error catastrophe limit, as observed before in cell culture studies. Finally, a model is proposed that correlates the mutagenicity of KP1212 with its tautomeric distribution in solution.

The folding mechanism of the N-terminal domain of ribosomal protein L9 (NTL91–39) is studied using temperature-jump (T-jump) amide I′ two-dimensional infrared (2D IR) spectroscopy in combination with spectral simulations based on a Markov state model (MSM) built from millisecond-long molecular dynamics trajectories. The results provide evidence for a compact well-structured folded state and a heterogeneous fast-exchanging denatured state ensemble exhibiting residual secondary structure. The folding rate of 26.4 μs−1 (at 80°C), extracted from the T-jump response of NTL91–39, compares favorably with the 18 μs−1 obtained from the MSM. Structural decomposition of the MSM and analysis along the folding coordinate indicates that helix-formation nucleates the global folding. Simulated difference spectra, corresponding to the global folding transition of the MSM, are in qualitative agreement with measured T-jump 2D IR spectra. The experiments demonstrate the use of T-jump 2D IR spectroscopy as a valuable tool for studying protein folding, with direct connections to simulations. The results suggest that in addition to predicting the correct native structure and folding time constant, molecular dynamics simulations carried out with modern force fields provide an accurate description of folding mechanisms in small proteins.

Structural diversification of canonical nucleic acid bases and nucleotide analogues by tautomerism has been proposed to be a powerful on/off switching mechanism allowing regulation of many biological processes mediated by RNA enzymes and aptamers. Despite the suspected biological importance of tautomerism, attempts to observe minor tautomeric forms in nucleic acid or hybrid nucleic acid-ligand complexes have met with challenges due to the lack of sensitive methods. Here, a combination of spectroscopic, biochemical, and computational tools probed tautomerism in the context of an RNA aptamer-ligand complex; studies involved a model ligand, oxythiamine pyrophosphate (OxyTPP), bound to the thiamine pyrophosphate (TPP) riboswitch (an RNA aptamer) as well as its unbound nonphosphorylated form, oxythiamine (OxyT). OxyTPP, similarly to canonical heteroaromatic nucleic acid bases, has a pyrimidine ring that forms hydrogen bonding interactions with the riboswitch. Tautomerism was established using two-dimensional infrared (2D IR) spectroscopy, variable temperature FTIR and NMR spectroscopies, binding isotope effects (BIEs), and computational methods. All three possible tautomers of OxyT, including the minor enol tautomer, were directly identified, and their distributions were quantitated. In the bound form, BIE data suggested that OxyTPP existed as a 4'-keto tautomer that was likely protonated at the N1'-position. These results also provide a mechanistic framework for understanding the activation of riboswitch in response to deamination of the active form of vitamin B1 (or TPP). The combination of methods reported here revealing the fine details of tautomerism can be applied to other systems where the importance of tautomerism is suspected.

We provide a systematic characterization of the nanosecond ground-state lactam–lactim tautomerization of pyridone derivatives in aqueous solution under ambient conditions using temperature-jump transient 2D IR spectroscopy. Although electronic excited-state tautomerization has been widely studied, experimental work on the ground electronic state, most relevant to chemistry and biology, is lacking. Using 2D IR spectroscopy, lactam and lactim tautomers of 6-chloro-2-pyridone and 2-chloro-4-pyridone are unambiguously identified by their unique cross-peak patterns. Monitoring the correlated exponential relaxation of these signals in response to a laser temperature jump provides a direct measurement of the nanosecond tautomerization kinetics. By studying the temperature, concentration, solvent, and pH dependence, we extract a thermodynamic and kinetic characterization and conclude that the tautomerization proceeds through a two-state concerted mechanism. We find that the intramolecular proton transfer is mediated by bridging water molecules and the reaction barrier is dictated by the release of a proton from pyridone, as would be expected for an efficient Grothuss-type proton transfer mechanism.

We provide a time- and structure-resolved characterization of the folding of the heterogeneous β-hairpin peptide Tryptophan Zipper 2 (Trpzip2) using 2D IR spectroscopy. The amide I′ vibrations of three Trpzip2 isotopologues are used as a local probe of the midstrand contacts, β-turn, and overall β-sheet content. Our experiments distinguish between a folded state with a type I′ β-turn and a misfolded state with a bulged turn, providing evidence for distinct conformations of the peptide backbone. Transient 2D IR spectroscopy at 45 °C following a laser temperature jump tracks the nanosecond and microsecond kinetics of unfolding and the exchange between conformers. Hydrogen bonds to the peptide backbone are loosened rapidly compared with the 5-ns temperature jump. Subsequently, all relaxation kinetics are characterized by an observed 1.2 ± 0.2-μs exponential. Our time-dependent 2D IR spectra are explained in terms of folding of either native or nonnative contacts from a common compact disordered state. Conversion from the disordered state to the folded state is consistent with a zip-out folding mechanism.

Two-dimensional infrared (2DIR) spectroscopy is a newly-developed experimental technique that measures protein structure and dynamics in solution with subpicosecond time resolution. Amide-I vibrations, consisting mainly of backbone C=O stretching modes, contain a wealth of structural information. Two-dimensional spectroscopy offers enhanced structural sensitivity by spreading the spectral information onto two frequency axes. Through a combination of temperature-jump 2DIR spectroscopy, isotope labeling, and Markov state models derived from molecular dynamics simulations, we develop a new method which can directly probe the structural rearrangements on timescales from nanoseconds to milliseconds. Markov state models provide an intuitive interpretation of the protein folding process while retaining much of the structural heterogeneity and diversity of folding pathways.The unfolding mechanism of a 39-residue α/β mini protein, NTL9, a two-state folder, is studied on timescales from 100 ns to 50 milliseconds. Transient 2DIR reveal a rapid sub-100 ns response that is attributed to weakening of the hydrogen-bonds, followed by unraveling of the beta sheet. The more stable helix is seen to denature on the 150 microsecond timescale. Experimental data is interpreted in the context of the recently-available Markov state model of NTL9. Simulated 2DIR spectra are generated for the structural ensemble, and are observed to be in great agreement with the temperature-jump 2DIR experiments. The results provide an elegant illustration of how a combination of cutting-edge experiments and state-of-the-art simulations gives new insights into the complex mechanism of protein folding.

We present a method to quantitatively determine the secondary structure composition of globular proteins using coherent two-dimensional infrared (2DIR) spectroscopy of backbone amide I vibrations (1550–1720 cm−1). Sixteen proteins with known crystal structures were used to construct a library of 2DIR spectra, and the fraction of residues in α-helix, β-sheet, and unassigned conformations was determined by singular value decomposition (SVD) of the measured two-dimensional spectra. The method was benchmarked by removing each individual protein from the set and comparing the composition extracted from 2DIR against the composition determined from the crystal structures. To highlight the increased structural content extracted from 2DIR spectra a similar analysis was also carried out using conventional infrared absorption of the proteins in the library., National Science Foundation (U.S.) (CHE-0616575), National Institutes of Health (U.S.) (CHE-0911107), Agilent Technologies

Polarization-dependent two-dimensional infrared (2D IR) spectra of the purine and pyrimadine base vibrations of five nucleotide monophosphates (NMPs) were acquired in D(2)O at neutral pH in the frequency range 1500-1700 cm(-1). The distinctive cross-peaks between the ring deformations and carbonyl stretches of NMPs indicate that these vibrational modes are highly coupled, in contrast with the traditional peak assignment, which is based on a simple local mode picture such as C═O, C═N, and C═C double bond stretches. A model of multiple anharmonically coupled oscillators was employed to characterize the transition energies, vibrational anharmonicities and couplings, and transition dipole strengths and orientations. No simple or intuitive structural correlations are found to readily assign the spectral features, except in the case of guanine and cytosine, which contain a single local CO stretching mode. To help interpret the nature of these vibrational modes, we performed density functional theory (DFT) calculations and found that multiple ring vibrations are coupled and delocalized over the purine and pyrimidine rings. Generally, there is close correspondence between the experimental and computational results, provided that the DFT calculations include explicit waters solvating hydrogen-bonding sites. These results provide direct experimental evidence of the delocalized nature of the nucleotide base vibrations via a nonperturbative fashion and will serve as building blocks for constructing a structure-based model of DNA and RNA vibrational spectroscopy.

Contains fulltext : 87660.pdf (Publisher’s version ) (Closed access) Isotope-edited two-dimensional infrared spectroscopy has been used to characterize the conformational heterogeneity of the beta-hairpin peptide TrpZip2 (TZ2) across its thermal unfolding transition. Four isotopologues were synthesized to probe hydrogen bonding and solvent exposure of the beta-turn (K8), the N-terminus (S1), and the midstrand region (T10 and T3T10). Isotope-shifts, 2D lineshapes, and other spectral changes to the amide I 2D IR spectra of labeled TZ2 isotopologues were observed as a function of temperature. Data were interpreted on the basis of structure-based spectroscopic modeling of conformers obtained from extensive molecular dynamics simulations. The K8 spectra reveal two unique turn geometries, the type I' beta-turn observed in the NMR structure, and a less populated disordered or bulged loop. The data indicate that structures at low temperature resemble the folded NMR structure with typical cross-strand hydrogen bonds, although with a subpopulation of misformed turns. As the temperature is raised from 25 to 85 degrees C, the fraction of population with a type I' turn increases, but the termini also fray. Hydrogen bonding contacts in the midstrand region remain at all temperatures although with increasing thermal disorder. Our data show no evidence of an extended chain or random coil state for the TZ2 peptide at any temperature. The methods demonstrated here offer an approach to characterizing conformational variation within the folded or unfolded states of proteins and peptides.

Multidimensional spectroscopies provide increased spectral information but time resolution is often limited by the picosecond lifetimes of the transitions they probe. At the expense of additional complexity, transient multidimensional techniques extend the accessible timescales for studying nonequilibrium chemical and biophysical phenomena. Transient temperature-jump (T-jump) experiments are particularly versatile, since they can be applied to any temperature-dependent change of state. We have developed a method to correct transient nonlinear techniques for distortions resulting from transient linear absorption of the probing pulses, distortions which can lead to false interpretations of the data. We apply these corrections in the collection of T-jump transient two-dimensional infrared spectra for the peptides diglycine and the β-hairpin peptide trpzip2. For diglycine, the T-jump induces changes in H-bonding, a response which is inherent to all aqueous systems. The trpzip2 results probe the hairpin unfolding kinetics and reveal two time scales

A novel class of antiviral drugs has been developed based on a principle termed “lethal mutagenesis” which aims at artificially increasing the mutation rates such that the viral population collapsed due to the intolerable amount of mutations. Specifically, KP1212 is a mutagenic deoxycytidine analog that elevates the G to A mutations in viral genome shown by clinical studies. It was hypothesized that the mutation originates from the tautomerization of KP1212 from the canonical amino-keto form to the imino-keto form, which results in favorable base pairing to A. We systematically characterized the tautomeric equilibria of KP1212 under physiological conditions using two-dimensional infrared (2D IR) spectroscopy. The intrinsic picosecond time-resolution of 2D IR enables us to detect various tautomers that are rapidly interconverting. Moreover, the unique 2D IR cross-peak patterns for each tautomer allow for unambiguous assignment which is not possible by traditional techniques. Strikingly, we observed significant amount of enol population which is regarded as the “rare” tautomer in DNA bases. Finally, we have performed temperature-jump (T-jump) 2D IR experiments to measure the tautomerization kinetics. We found that with a 10oC T-jump, the enol populations increase at the expense of the keto populations on the nanosecond timescale. The nanosecond tautomerization is much faster than the speed of polymerase (1 millisecond/base), therefore allowing sufficient time for the formation of mismatched base pairs.