UV/Visible Diffusion-Ordered Spectroscopy: A Simultaneous Probe of Molecular Size and Electronic Absorption.

Based on concepts from nuclear magnetic resonance, we have developed UV/vis diffusion-ordered spectroscopy, which simultaneously probes the size and electronic absorption spectrum of molecules and particles. We use simple flow technology to create a step-function concentration profile inside an optical sample cell, and by measuring the time-dependent absorption spectrum in an initially solvent-filled part of the sample volume, we obtain the diffusion coefficients and UV/vis spectra of the species present in the sample solution. From these data, we construct a two-dimensional spectrum with absorption wavelength on one axis and diffusion coefficient (or equivalently, size) on the other, in which the UV/vis spectrum of a mixture with different molecular sizes is separated into the spectra of the different species, sorted by size.

Elucidating the role of water in collagen self-assembly by isotopically modulating collagen hydration.

Water is known to play an important role in collagen self-assembly, but it is still largely unclear how water-collagen interactions influence the assembly process and determine the fibril network properties. Here, we use the H[Formula: see text]O/D[Formula: see text]O isotope effect on the hydrogen-bond strength in water to investigate the role of hydration in collagen self-assembly. We dissolve collagen in H[Formula: see text]O and D[Formula: see text]O and compare the growth kinetics and the structure of the collagen assemblies formed in these water isotopomers.

DO as an Imperfect Replacement for HO: Problem or Opportunity for Protein Research?

DO is commonly used as a solvent instead of HO in spectroscopic studies of proteins, in particular, in infrared and nuclear-magnetic-resonance spectroscopy. DO is chemically equivalent to HO, and the differences, particularly in hydrogen-bond strength, are often ignored. However, replacing solvent water with DO can affect not only the kinetics but also the structure and stability of biomolecules.

Raman Diffusion-Ordered Spectroscopy.

The Stokes-Einstein relation, which relates the diffusion coefficient of a molecule to its hydrodynamic radius, is commonly used to determine molecular sizes in chemical analysis methods. Here, we combine the size sensitivity of such diffusion-based methods with the structure sensitivity of Raman spectroscopy by performing Raman diffusion-ordered spectroscopy (Raman-DOSY). The core of the Raman-DOSY setup is a flow cell with a Y-shaped channel containing two inlets: one for the sample solution and one for the pure solvent.

Multidimensional infrared diffusion-ordered spectroscopy in depletion mode distinguishes protein amyloids and monomers.

Conventional and two-dimensional infrared (2D-IR) spectroscopy are well suited to study amyloid aggregates, because the amide I mode is a sensitive probe of the aggregate structure. However, these methods are not so useful to study mixtures of aggregates and monomers, which generally have overlapping amide I spectra. Here, we show that IR-Diffusion-Ordered Spectroscopy can disentangle the contributions of protein monomers and aggregates (amyloids) in FTIR and 2D-IR spectra by separating the spectral contributions based on molecular size.

Hydrogen Bonds under Stress: Strain-Induced Structural Changes in Polyurethane Revealed by Rheological Two-Dimensional Infrared Spectroscopy.

The remarkable elastic properties of polymers are ultimately due to their molecular structure, but the relation between the macroscopic and molecular properties is often difficult to establish, in particular for (bio)polymers that contain hydrogen bonds, which can easily rearrange upon mechanical deformation. Here we show that two-dimensional infrared spectroscopy on polymer films in a miniature stress tester sheds new light on how the hydrogen-bond structure of a polymer is related to its viscoelastic response. We study thermoplastic polyurethane, a block copolymer consisting of hard segments of hydrogen-bonded urethane groups embedded in a soft matrix of polyether chains.

In Situ Identification of Secondary Structures in Unpurified Silk Fibrils Using Polarized Two-Dimensional Infrared Spectroscopy.

The mechanical properties of biomaterials are dictated by the interactions and conformations of their building blocks, typically proteins. Although the macroscopic behavior of biomaterials is widely studied, our understanding of the underlying molecular properties is generally limited. Among the noninvasive and label-free methods to investigate molecular structures, infrared spectroscopy is one of the most commonly used tools because the absorption bands of amide groups strongly depend on protein secondary structure.

Infrared Diffusion-Ordered Spectroscopy Reveals Molecular Size and Structure.

Inspired by ideas from NMR, we have developed Infrared Diffusion-Ordered Spectroscopy (IR-DOSY), which simultaneously characterizes molecular structure and size. We rely on the fact that the diffusion coefficient of a molecule is determined by its size through the Stokes-Einstein relation, and achieve sensitivity to the diffusion coefficient by creating a concentration gradient and tracking its equilibration in an IR-frequency resolved manner. Analogous to NMR-DOSY, a two-dimensional IR-DOSY spectrum has IR frequency along one axis and diffusion coefficient (or equivalently, size) along the other, so the chemical structure and the size of a compound are characterized simultaneously.

Broadband Multidimensional Spectroscopy Identifies the Amide II Vibrations in Silkworm Films.

We used two-dimensional infrared spectroscopy to disentangle the broad infrared band in the amide II vibrational regions of native silk films, identifying the single amide II modes and correlating them to specific secondary structure. Amide I and amide II modes have a strong vibrational coupling, which manifests as cross-peaks in 2D infrared spectra with frequencies determined by both the amide I and amide II frequencies of the same secondary structure. By cross referencing with well-known amide I assignments, we determined that the amide II (N-H) absorbs at around 1552 and at 1530 cm for helical and β-sheet structures, respectively.

Molecular Structure and Surface Accumulation Dynamics of Hyaluronan at the Water-Air Interface.

Hyaluronan is a biopolymer that is essential for many biological processes in the human body, like the regulation of tissue lubrication and inflammatory responses. Here, we study the behavior of hyaluronan at aqueous surfaces using heterodyne-detected vibrational sum-frequency generation spectroscopy (HD-VSFG). Low-molecular-weight hyaluronan (∼150 kDa) gradually covers the water-air interface within hours, leading to a negatively charged surface and a reorientation of interfacial water molecules.

Disaccharide Residues are Required for Native Antifreeze Glycoprotein Activity.

Antifreeze glycoproteins (AFGPs) are able to bind to ice, halt its growth, and are the most potent inhibitors of ice recrystallization known. The structural basis for AFGP's unique properties remains largely elusive. Here we determined the antifreeze activities of AFGP variants that we constructed by chemically modifying the hydroxyl groups of the disaccharide of natural AFGPs.

Strong Reduction of the Chain Rigidity of Hyaluronan by Selective Binding of Ca Ions.

The biological functions of natural polyelectrolytes are strongly influenced by the presence of ions, which bind to the polymer chains and thereby modify their properties. Although the biological impact of such modifications is well recognized, a detailed molecular picture of the binding process and of the mechanisms that drive the subsequent structural changes in the polymer is lacking. Here, we study the molecular mechanism of the condensation of calcium, a divalent cation, on hyaluronan, a ubiquitous polymer in human tissues.

Connecting the Stimuli-Responsive Rheology of Biopolymer Hydrogels to Underlying Hydrogen-Bonding Interactions.

Many biopolymer hydrogels are environmentally responsive because they are held together by physical associations that depend on pH and temperature. Here, we investigate how the pH and temperature responses of the rheology of hyaluronan hydrogels are connected to the underlying molecular interactions. Hyaluronan is an essential structural biopolymer in the human body with many applications in biomedicine.

Effect of intramolecular hydrogen-bond formation on the molecular conformation of amino acids.

The molecular conformation of the carboxyl group can be crucial for its chemical properties and intermolecular interactions, especially in complex molecular environments such as polypeptides. Here, we study the conformational behaviour of the model amino acid N-acetylproline in solution at room temperature with two-dimensional infrared spectroscopy. We find that the carboxyl group of N-acetylproline adopts two distinct conformations, syn- and anti-.

Peptide Side-COOH Groups Have Two Distinct Conformations under Biorelevant Conditions.

The carboxyl (COOH) side chain groups of amino acids, such as aspartic acid, play an important role in biochemical processes, including enzymatic proton transport. In many theoretical studies, it was found that the (bio)chemical reactivity of the carboxyl group strongly depends on the conformation of this group. Interestingly, up to now there has been no experimental investigation of the geometry and the stability of different COOH conformers under biorelevant conditions.

Hyaluronan biopolymers release water upon pH-induced gelation.

We study the relation between the macroscopic viscoelastic properties of aqueous hyaluronan polymer solutions and the molecular-scale dynamics of water using rheology measurements, differential dynamic microscopy, and polarization-resolved infrared pump-probe spectroscopy. We observe that the addition of hyaluronan to water leads to a slowing down of the reorientation of a fraction of the water molecules. Near pH 2.

Direct Observation of Intrachain Hydrogen Bonds in Aqueous Hyaluronan.

We use two-dimensional infrared spectroscopy to study the interactions between the amide and carboxylate anion groups of hyaluronan polymers at neutral pH. The spectra reveal the presence of intrachain hydrogen bonds between the amide and carboxylate anion groups in aqueous solution. We determine the relative orientation of the amide and carboxylate anion groups when forming this hydrogen bond and quantify the fraction of amide groups that participate in hydrogen bonding.

Observation of Distinct Carboxylic Acid Conformers in Aqueous Solution.

We investigate the molecular geometry of the carboxyl group of formic acid in acetonitrile and aqueous solutions at room temperature with two-dimensional infrared spectroscopy (2D-IR). We found that the carboxyl group adopts two distinct configurations: a configuration in which the carbonyl group is oriented antiparallel to the hydroxyl (anti-conformer), and a configuration in which the carbonyl group is oriented at an angle of ∼60° with respect to the hydroxyl (syn-conformer). These results constitute the first experimental evidence that carboxyl groups exist as two distinct and long-living conformational isomers in aqueous solution at room temperature.

Molecular Origin of the Elastic State of Aqueous Hyaluronic Acid.

The macroscopic mechanical properties of biological hydrogels are broadly studied and successfully mimicked in synthetic materials, but little is known about the molecular interactions that mediate these properties. Here, we use two-dimensional infrared spectroscopy to study the pH-induced gelation of hyaluronic acid, a ubiquitous biopolymer, which undergoes a transition from a viscous to an elastic state in a narrow pH range around 2.5.

Determination of the Solution Structure of Antifreeze Glycoproteins Using Two-Dimensional Infrared Spectroscopy.

We study the solution structure of antifreeze glycoproteins (AFGPs) with linear and two-dimensional infrared spectroscopy (2D-IR). With 2D-IR, we study the coupling between the amide I and amide II vibrations of AFGPs. The measured nonlinear spectral response constitutes a much more clearly resolved amide I spectrum than the linear absorption spectrum of the amide I vibrations and allows us to identify the different structural elements of AFGPs in solution.

Rotational Cation Dynamics in Metal Halide Perovskites: Effect on Phonons and Material Properties.

The dynamics of organic cations in metal halide hybrid perovskites (MHPs) have been investigated using numerous experimental and computational techniques because of their suspected effects on the properties of MHPs. In this Perspective, we summarize and reconcile key findings and present new data to synthesize a unified understanding of the dynamics of the cations. We conclude that theory and experiment collectively paint a relatively complete picture of rotational dynamics within MHPs.

Strategies To Increase the Thermal Stability of Truly Biomimetic Hydrogels: Combining Hydrophobicity and Directed Hydrogen Bonding.

Enhancing the thermal stability of proteins is an important task for protein engineering. There are several ways to increase the thermal stability of proteins in biology, such as greater hydrophobic interactions, increased helical content, decreased occurrence of thermolabile residues, or stable hydrogen bonds. Here, we describe a well-defined polymer based on β-helical polyisocyanotripeptides (TriPIC) that uses biological approaches, including hydrogen bonding and hydrophobic interactions for its exceptional thermal stability in aqueous solutions.