Ayanjeet Ghosh

Tidal surge in the M2 proton channel, sensed by 2D IR spectroscopy

The M2 proton channel from influenza A virus transmits protons across membranes via a narrow aqueous pore lined by water and a proton sensor, His37. Near the center of the membrane, a water cluster is stabilized by the carbonyl of Gly34 and His37, the properties of which are modulated by protonation of His37. At low pH (5–6), where M2 conducts protons, this region undergoes exchange processes on the microsecond to second timescale. Here, we use 2D IR to examine the instantaneous conformational distribution and hydration of G34, and the evolution of the ensemble on the femtosecond to picosecond timescale. The channel water is strongly pH dependent as gauged by 2D IR which allows recording of the vibrational frequency autocorrelation function of a 13C = 18O Gly34 probe. At pH 8, where entry and exit of protons within the channel are very slow, the carbonyl groups appear to adopt a single conformation/environment. The high-pH conformer does not exhibit spectral dynamics near the Gly34, and water in the channel must form a relatively rigid ice-like structure. By contrast, two vibrational forms of G34 are seen at pH 6.2, neither of which is identical to the high-pH form. In at least one of these low-pH forms, the probe is immersed in a very mobile, bulk-like aqueous environment having a correlation time ca. 1.3 ps at pH 6.2. Thus, protonation of His37 at low pH causes liquid-like water molecules to flow into the neighborhood of the Gly34.

Ester Carbonyl Vibration as a Sensitive Probe of Protein Local Electric Field

The ability to quantify the local electrostatic environment of proteins and protein/peptide assemblies is key to gaining a microscopic understanding of many biological interactions and processes. Herein, we show that the ester carbonyl stretching vibration of two non-natural amino acids, L-aspartic acid 4-methyl ester and L-glutamic acid 5-methyl ester, is a convenient and sensitive probe in this regard, since its frequency correlates linearly with the local electrostatic field for both hydrogen-bonding and non-hydrogen-bonding environments. We expect that the resultant frequency-electric-field map will find use in various applications. Furthermore, we show that, when situated in a non-hydrogen-bonding environment, this probe can also be used to measure the local dielectric constant (ε). For example, its application to amyloid fibrils formed by Aβ(16-22) revealed that the interior of such β-sheet assemblies has an εu2005value of approximately 5.6.

Identification of Arginine Residues in Peptides by 2D-IR Echo Spectroscopy

The CN stretching vibrations of the guanidyl group in the arginine dipeptide side chain are examined by two-dimensional infrared spectroscopy. In D(2)O, the spectra display two distinct diagonal peaks. These nearly degenerate modes undergo ultrafast energy transfer. The energy-transfer rate was determined directly from the 2D-IR spectra to be 1/2.1 ps(-1). The cross peaks in 2D-IR arising from the energy transfer provide a definitive identification of arginine in larger proteins. An example of arginine in the transmembrane protein M2, found in influenza viruses, is given.

2D IR spectroscopy reveals the role of water in the binding of channel-blocking drugs to the influenza M2 channel

Water is an integral part of the homotetrameric M2 proton channel of the influenza A virus, which not only assists proton conduction but could also play an important role in stabilizing channel-blocking drugs. Herein, we employ two dimensional infrared (2D IR) spectroscopy and site-specific IR probes, i.e., the amide I bands arising from isotopically labeled Ala30 and Gly34 residues, to probe how binding of either rimantadine or 7,7-spiran amine affects the water dynamics inside the M2 channel. Our results show, at neutral pH where the channel is non-conducting, that drug binding leads to a significant increase in the mobility of the channel water. A similar trend is also observed at pH 5.0 although the difference becomes smaller. Taken together, these results indicate that the channel water facilitates drug binding by increasing its entropy. Furthermore, the 2D IR spectral signatures obtained for both probes under different conditions collectively support a binding mechanism whereby amantadine-like drugs dock in the channel with their ammonium moiety pointing toward the histidine residues and interacting with a nearby water cluster, as predicted by molecular dynamics simulations. We believe these findings have important implications for designing new anti-influenza drugs.

Watching Proteins Wiggle: Mapping Structures with Two-Dimensional Infrared Spectroscopy

Proteins exhibit structural fluctuations over decades of time scales. From the picosecond side chain motions to aggregates that form over the course of minutes, characterizing protein structure over these vast lengths of time is important to understanding their function. In the past 15 years, two-dimensional infrared spectroscopy (2D IR) has been established as a versatile tool that can uniquely probe proteins structures on many time scales. In this review, we present some of the basic principles behind 2D IR and show how they have, and can, impact the field of protein biophysics. We highlight experiments in which 2D IR spectroscopy has provided structural and dynamical data that would be difficult to obtain with more standard structural biology techniques. We also highlight technological developments in 2D IR that continue to expand the scope of scientific problems that can be accessed in the biomedical sciences.

Association of aureolic acid antibiotic, chromomycin A3 with Cu2+ and its negative effect upon DNA binding property of the antibiotic

Here we have examined the association of an aureolic acid antibiotic, chromomycin A3 (CHR), with Cu2+. CHR forms a high affinity 2:1 (CHR:Cu2+) complex with dissociation constant of 0.08xa0×xa010−10xa0M2 at 25°C, pH 8.0. The affinity of CHR for Cu2+ is higher than those for Mg2+ and Zn2+ reported earlier from our laboratory. CHR binds preferentially to Cu2+ in presence of equimolar amount of Zn2+. Complex formation between CHR and Cu2+ is an entropy driven endothermic process. Difference between calorimetric and van’t Hoff enthalpies indicate the presence of multiple equilibria, supported from biphasic nature of the kinetics of association. Circular dichroism spectroscopy show that [(CHR)2:Cu2+] complex assumes a structure different from either of the Mg2+ and Zn2+ complex reported earlier. Both [(CHR)2:Mg2+] and [(CHR)2:Zn2+] complexes are known to bind DNA. In contrast, [(CHR)2:Cu2+] complex does not interact with double helical DNA, verified by means of Isothermal Titration Calorimetry of its association with calf thymus DNA and the double stranded decamer (5′-CCGGCGCCGG-3′). In order to interact with double helical DNA, the (antibiotic)2 : metal (Mg2+ and Zn2+) complexes require a isohelical conformation. Nuclear Magnetic Resonance spectroscopy shows that the Cu2+ complex adopts a distorted octahedral structure, which cannot assume the required conformation to bind to the DNA. This report demonstrates the negative effect of a bivalent metal upon the DNA binding property of CHR, which otherwise binds to DNA in presence of metals like Mg2+and Zn2+. The results also indicate that CHR has a potential for chelation therapy in Cu2+ accumulation diseases. However cytotoxicity of the antibiotic might restrict the use.

Experimental implementations of 2D IR spectroscopy through a horizontal pulse shaper design and a focal plane array detector

Aided by advances in optical engineering, two-dimensional infrared spectroscopy (2D IR) has developed into a promising method for probing structural dynamics in biophysics and material science. We report two new advances for 2D IR spectrometers. First, we report a fully reflective and totally horizontal pulse shaper, which significantly simplifies alignment. Second, we demonstrate the applicability of mid-IR focal plane arrays (FPAs) as suitable detectors in 2D IR experiments. FPAs have more pixels than conventional linear arrays and can be used to multiplex optical detection. We simultaneously measure the spectra of a reference beam, which improves the signal-to-noise by a factor of 4; and two additional beams that are orthogonally polarized probe pulses for 2D IR anisotropy experiments.

2D IR spectroscopy of histidine: probing side-chain structure and dynamics via backbone amide vibrations.

It is well known that histidine is involved in many biological functions due to the structural versatility of its side chain. However, probing the conformational transitions of histidine in proteins, especially those occurring on an ultrafast time scale, is difficult. Herein we show, using a histidine dipeptide as a model, that it is possible to probe the tautomer and protonation status of a histidine residue by measuring the two-dimensional infrared (2D IR) spectrum of its amide I vibrational transition. Specifically, for the histidine dipeptide studied, the amide unit of the histidine gives rise to three spectrally resolvable amide I features at approximately 1630, 1644, and 1656 cm–1, respectively, which, based on measurements at different pH values and frequency calculations, are assigned to a τ tautomer (1630 cm–1 component) and a π tautomer with a hydrated (1644 cm–1 component) or dehydrated (1656 cm–1 component) amide. Because of the intrinsic ultrafast time resolution of 2D IR spectroscopy, we believe that the current approach, when combined with the isotope editing techniques, will be useful in revealing the structural dynamics of key histidine residues in proteins that are important for function.

Wide-field FTIR microscopy using mid-IR pulse shaping

We have developed a new table-top technique for collecting wide-field Fourier transform infrared (FTIR) microscopic images by combining a femtosecond pulse shaper with a mid-IR focal plane array. The pulse shaper scans the delay between a pulse pair extremely rapidly for high signal-to-noise, while also enabling phase control of the individual pulses to under-sample the interferograms and subtract background. Infrared absorption images were collected for a mixture of W(CO)₆ or Mn₂(CO)₁₀ absorbed polystyrene beads, demonstrating that this technique can spatially resolve chemically distinct species. The images are sub-diffraction limited, as measured with a USAF test target patterned on CaF₂ and verified with scalar wave simulations. We also find that refractive, rather than reflective, objectives are preferable for imaging with coherent radiation. We discuss this method with respect to conventional FTIR microscopes.

Structural Characterization of Single-Stranded DNA Monolayers Using Two-Dimensional Sum Frequency Generation Spectroscopy

DNA-covered materials are important in technological applications such as biosensors and microarrays, but obtaining structural information on surface-bound biomolecules is experimentally challenging. In this paper, we structurally characterize single-stranded DNA monolayers of poly(thymine) from 10 to 25 bases in length with an emerging surface technique called two-dimensional sum frequency generation (2D SFG) spectroscopy. These experiments are carried out by adding a mid-IR pulse shaper to a femtosecond broad-band SFG spectrometer. Cross peaks and 2D line shapes in the 2D SFG spectra provide information about structure and dynamics. Because the 2D SFG spectra are heterodyne detected, the monolayer spectra can be directly compared to 2D infrared (2D IR) spectra of poly(thymine) in solution, which aids interpretation. We simulate the 2D SFG spectra using DFT calculations and an excitonic Hamiltonian that relates the molecular geometry to the vibrational coupling. Intrabase cross peaks help define the orientation of the bases and interbase cross peaks, created by coupling between bases, and resolves features not observed in 1D SFG spectra that constrain the relative geometries of stacked bases. We present a structure for the poly(T) oligomer that is consistent with the 2D SFG data. These experiments provide insight into the DNA monolayer structure and set precedent for studying complex biomolecules on surfaces with 2D SFG spectroscopy.