Evidence for Coupling between Nitrile Groups Using DNA Templates: A Promising New Method for Monitoring Structures with Infrared Spectroscopy

Infrared spectroscopy is a common method for monitoring biomolecular structures but suffers from spectral congestion. Non-natural vibrational probes provide a way to regain structural specificity because they provide a unique vibrational signature and can be incorporated into proteins or other biomolecules at specific locations. A popular probe is the nitrile group because its frequency is sensitive to the electrostatics of its environment. In this work, we show that pairs of nitrile groups can be used to directly probe distances and angles in dual labeled molecules. By labeling model DNA oligomers with pairs of nitrile tags, we demonstrate that the vibrational coupling between two nitrile groups is strong enough that Fourier transform infrared (FTIR) spectra can be used to probe relative nitrile distances >4.5 Å. Our approach is similar in spirit to monitoring structures with fluorescence resonance energy transfer (FRET) using a pair of fluorescent labels or a pair of spin labels in electron spin resonance spectroscopy. The small sizes of nitrile groups make especially valuable probes of sterically confined regions like the inner cores of large biomolecules where other spectroscopic probes do not fit

A Tribute to Daniel M. Neumark

A Tribute to Daniel M. Neumar

Extracting Structural Information from the Polarization Dependence of One- and Two-Dimensional Sum Frequency Generation Spectra

We present ways in which pulse sequences and polarizations can be used to extract structural information from one- and two-dimensional vibrational sum frequency generation (2D SFG) spectra. We derive analytic expressions for the polarization dependence of systems containing coupled vibrational modes, and we present simulated spectra to identify the features of different molecular geometries. We discuss several useful polarization combinations for suppressing strong diagonal peaks and emphasizing weaker cross-peaks. We investigate unique capabilities of 2D SFG spectra for obtaining structural information about SFG-inactive modes and for identifying coupled achiral chromophores. This work builds on techniques that have been developed for extracting structural information from 2D IR spectra. This paper discusses how to utilize these concepts in 2D SFG experiments to probe multioscillator systems at interfaces. The sample code for calculating polarization dependence of 1D and 2D SFG spectra is provided in the Supporting Information

Ultrafast Fluctuations in PM6 Domains of Binary and Ternary Organic Photovoltaic Thin Films Probed with Two-Dimensional White-Light Spectroscopy

We present two-dimensional white-light spectroscopy (2DWL) measurements of binary and ternary bulk heterojunctions of the polymer donor PM6 mixed with state-of-the-art nonfullerene acceptors Y6 or IT4F. The ternary film has a shorter lifetime and faster spectral diffusion than either of the binary films. 2D line shape analysis of the PM6 ground state bleach with a Kubo model determines that all three films have similar amplitudes of fluctuations (Δ = 0.29 fs–1) in their transition frequencies, but different relaxation times (ranging from 102 to 24 fs). The ternary film exhibits faster dynamics than either of the binary films. The short lifetime of the ternary blend is consistent with increased photoexcitation transfer and the fast frequency fluctuations are consistent with structural dynamics of aliphatic side chains. These results suggest that the femtosecond fluctuations of PM6 are impacted by the choice of the acceptor molecules. We hypothesize that those dynamics are either indicative, or perhaps the initial source, of structural dynamics that ultimately contribute to solar cell operation

Transition Dipoles from 1D and 2D Infrared Spectroscopy Help Reveal the Secondary Structures of Proteins: Application to Amyloids

Transition dipoles are an underutilized quantity for probing molecular structures. The transition dipole strengths in an extended system like a protein are modulated by the couplings and thus probe the structures. Here we measure the absolute transition dipole strengths of human and rat amylin in their solution, aggregated, membrane, and micelleular bound forms, using a combination of 1D and 2D infrared spectroscopy. We find that the vibrational modes of amyloid fibers made of human amylin can extend across as many as 12 amino acids, reflecting very ordered β-sheets in the most carefully prepared samples. Rat amylin has FTIR spectra that are nearly identical in solution, micelles, and membranes. We show that the transition dipoles of rat amylin are much larger when bound to micelles and membranes than when in solution, consistent with rat amylin adopting an α-helical structure. We interpret the transition dipole strengths as experimental measurements of the inverse participation ratio often calculated in theoretical studies. The structure of aggregating and membrane-bound proteins can be difficult to identify with existing techniques, especially during kinetics. These results demonstrate how absolute transition dipoles measured via our 1D/2D spectroscopy method can provide important structural information

Time-Domain Photothermal AFM Spectroscopy via Femtosecond Pulse Shaping

A time-domain version of photothermal microscopy using an atomic force microscope (AFM) is reported, which we call Fourier transform photothermal (FTPT) spectroscopy, where the delay between two laser pulses is varied and the Fourier transform is computed. An acousto-optic modulator-based pulse shaper sets the delay and phases of the pulses shot-to-shot at 100 kHz, enabling background subtraction and data collection in the rotating frame. The pulse shaper is also used to flatten the pulse spectrum, thereby eliminating the need for normalization by the laser spectrum. We demonstrate the method on 6,13-bis(triisopropylsilylethynyl)pentacene (TIPS-Pn) microcrystals and Mn-phthalocyanine islands, confirming subdiffraction spatial resolution, and providing new spectroscopic insights likely linked to structural defects in the crystals

Structural and Sequence Analysis of the Human γD-Crystallin Amyloid Fibril Core Using 2D IR Spectroscopy, Segmental ¹³C Labeling, and Mass Spectrometry

Identifying the sequence and structural content of residues that compose the core of amyloid fibrils is important because core regions likely control the process of fibril extension and provide potential drug targets. Human γD-crystallin is an eye lens protein that aggregates into amyloid fibrils under acidic conditions. In this manuscript, we use a pepsin enzymatic digest to isolate the core of the amyloid fibrils. The sequence of the core is identified with MALDI MS/MS and its structure is probed with 2D IR spectroscopy and ¹³C isotope labeling. Mass spectrometry of the digest identifies residues 80–163 as the amyloid core, which spans most of the C-terminal domain, the linker, and a small portion of the N-terminal domain. From 2D IR spectroscopy of the digested fibrils, we learn that only the C-terminal domain contributes to the amyloid β-sheets while the N-terminal and linker residues are disordered. A comparison to the native crystal structure reveals that loops and α-helices in the native state must undergo conformational transitions to β-strands upon aggregation. These locations may be good drug binding targets. Besides providing new information about γD -crystallin, this study demonstrates the complementarity of mass spectrometry and 2D IR spectroscopy to obtain both sequence and structure information that neither technique provides individually, which will be especially useful for samples only available in microgram quantities

Tracking Fiber Formation in Human Islet Amyloid Polypeptide with Automated 2D-IR Spectroscopy

Tracking Fiber Formation in Human Islet Amyloid Polypeptide with Automated 2D-IR Spectroscop

Solvent-Independent Anharmonicity for Carbonyl Oscillators

The physical origins of vibrational frequency shifts have been extensively studied in order to understand noncovalent intermolecular interactions in the condensed phase. In the case of carbonyls, vibrational solvatochromism, MD simulations, and vibrational Stark spectroscopy suggest that the frequency shifts observed in simple solvents arise predominately from the environment’s electric field due to the vibrational Stark effect. This is contrary to many previously invoked descriptions of vibrational frequency shifts, such as bond polarization, whereby the bond’s force constant and/or partial nuclear charges are altered due to the environment, often illustrated in terms of favored resonance structures. Here we test these hypotheses using vibrational solvatochromism as measured using 2D IR to assess the solvent dependence of the bond anharmonicity. These results indicate that the carbonyl bond’s anharmonicity is independent of solvent as tested using hexanes, DMSO, and D2O and is supported by simulated 2D spectra. In support of the linear vibrational Stark effect, these 2D IR measurements are consistent with the assertion that the Stark tuning rate is unperturbed by the electric field generated by both hydrogen and non-hydrogen bonding environments and further extends the general applicability of carbonyl probes for studying intermolecular interactions

Amyloid Fiber Formation in Human γD-Crystallin Induced by UV–B Photodamage

γD-Crystallin is an abundant structural protein of the lens that is found in native and modified forms in cataractous aggregates. We establish that UV–B irradiation of γD-Crystallin leads to structurally specific modifications and precipitation via two mechanisms: amorphous aggregates and amyloid fibers. UV–B radiation causes cleavage of the backbone, in large measure near the interdomain interface, where side chain oxidations are also concentrated. 2D IR spectroscopy and expressed protein ligation localize fiber formation exclusively to the C-terminal domain of γD-Crystallin. The native β-sandwich domains are not retained upon precipitation by either mechanism. The similarities between the amyloid forming pathways when induced by either UV–B radiation or low pH suggest that the propensity for the C-terminal β-sandwich domain to form amyloid β-sheets determines the misfolding pathway independent of the mechanism of denaturation