Katarzyna M. Tych

Terahertz vibrational absorption spectroscopy using microstrip-line waveguides

We demonstrate that terahertz microstrip-line waveguides can be used to measure absorption spectra of polycrystalline materials with a high frequency resolution (∼2 GHz) and with a spatial resolution that is determined by the microstrip-line dimensions, rather than the free-space wavelength. The evanescent terahertz-bandwidth electric field extending above the microstrip line interacts with, and is modified by, overlaid dielectric samples, thus enabling the characteristic vibrational absorption resonances in the sample to be probed. As an example, the terahertz absorption spectrum of polycrystalline lactose monohydrate was investigated; the lowest lying mode was observed at 534(±2) GHz, in excellent agreement with free-space measurements. This microstrip technique offers both a higher spatial and frequency resolution than free-space terahertz time-domain spectroscopy and requires no contact between the waveguide and sample.

Terahertz pulsed spectroscopic imaging using optimized binary masks

We report the development of a terahertz pulsed spectroscopic imaging system based on the concept of compressive sensing. A single-point terahertz detector, together with a set of 40 optimized two-dimensional binary masks, was used to measure the terahertz waveforms transmitted through a sample. Terahertz time- and frequency-domain images of the sample comprising 20×20 pixels were subsequently reconstructed. We demonstrate that both the spatial distribution and the spectral characteristics of a sample can be obtained by this means. Compared with conventional terahertz pulsed imaging, no raster scanning of the object is required, and ten times fewer terahertz spectra need be taken. It is therefore ideal for real-time imaging applications.

Single-Molecule Force Spectroscopy Identifies a Small Cold Shock Protein as Being Mechanically Robust

Single-molecule force spectroscopy has emerged as a powerful approach to examine the stability and dynamics of single proteins. We have completed force extension experiments on the small cold shock protein B from Thermotoga maritima, using a specially constructed chimeric polyprotein. The proteins simple topology, which is distinct from the mechanically well-characterized β-grasp and immunoglobulin (Ig)-like folds, in addition to the wide range of structural homologues resulting from its ancient origin, provides an attractive model protein for single-molecule force spectroscopy studies. We have determined that the protein has mechanical stability, unfolding at greater than 70 pN at a pulling velocity of 100 nm s(-1). We reveal features of the unfolding energy landscape by measuring the dependence of the mechanical stability on pulling velocity, in combination with Monte Carlo simulations. We show that the cold shock protein has mechanically robust, yet malleable, features that may be important in providing the protein with stability and flexibility to function over a range of environmental conditions. These results provide insights into the relationship between the secondary structure and topology of a protein and its mechanical strength. This lays the foundation for the investigation of the effects of changes in environmental conditions on the mechanical and dynamic properties of cold shock proteins.

Applying broadband terahertz time‐domain spectroscopy to the analysis of crystalline proteins: a dehydration study

The application of terahertz time-domain spectroscopy and imaging to the study of proteins in crystalline form is demonstrated. Terahertz time-domain spectroscopy is particularly sensitive to the long-range ordering of molecules, with proven utility for the spectroscopy of crystalline biological small molecules. Here, the terahertz time-domain absorption response of a macromolecular protein single crystal is investigated for the first time. In particular, the effect of dehydration on the terahertz absorption coefficient of tetragonal hen egg white lysozyme crystals is reported.

Rapid and Robust Polyprotein Production Facilitates Single-Molecule Mechanical Characterization of β-Barrel Assembly Machinery Polypeptide Transport Associated Domains.

Single-molecule force spectroscopy by atomic force microscopy exploits the use of multimeric protein constructs, namely, polyproteins, to decrease the impact of nonspecific interactions, to improve data accumulation, and to allow the accommodation of benchmarking reference domains within the construct. However, methods to generate such constructs are either time- and labor-intensive or lack control over the length or the domain sequence of the obtained construct. Here, we describe an approach that addresses both of these shortcomings that uses Gibson assembly (GA) to generate a defined recombinant polyprotein rapidly using linker sequences. To demonstrate the feasibility of this approach, we used GA to make a polyprotein composed of alternating domains of I27 and TmCsp, (I27-TmCsp)3-I27)(GA), and showed the mechanical fingerprint, mechanical strength, and pulling speed dependence are the same as an analogous polyprotein constructed using the classical approach. After this benchmarking, we exploited this approach to facilitiate the mechanical characterization of POTRA domain 2 of BamA from E. coli (EcPOTRA2) by assembling the polyprotein (I27-EcPOTRA2)3-I27(GA). We show that, as predicted from the α + β topology, EcPOTRA2 domains are mechanically robust over a wide range of pulling speeds. Furthermore, we identify a clear correlation between mechanical robustness and brittleness for a range of other α + β proteins that contain the structural feature of proximal terminal β-strands in parallel geometry. We thus demonstrate that the GA approach is a powerful tool, as it circumvents the usual time- and labor-intensive polyprotein production process and allows for rapid production of new constructs for single-molecule studies. As shown for EcPOTRA2, this approach allows the exploration of the mechanical properties of a greater number of proteins and their variants. This improves our understanding of the relationship between structure and mechanical strength, increasing our ability to design proteins with tailored mechanical properties.

Measurement and analysis of the diffuse reflectance of powdered samples at terahertz frequencies using a quantum cascade laser

We report terahertz (THz) diffuse reflectance measurements of bulk powdered samples at a frequency of 2.83 THz using a narrowband quantum cascade laser. Samples studied comprise polydisperse powders with absorption coefficients extending over two orders of magnitude from ∼3 cm(-1) to >200 cm(-1). Diffuse reflectance measurements are used to obtain the effective absorption coefficient of these samples from the backscattering cross-section, predicted under the quasi-crystalline approximation (QCA) in the T-matrix formulation and in conjunction with the Percus-Yevick pair distribution function. Results are compared with effective absorption coefficients obtained from THz time-domain spectroscopy measurements on pressed pellet samples, and show good agreement over the range of effective absorption coefficients studied. We observe that the backscattering cross-section predicted under the QCA is strongly dependent on both the real and imaginary components of the complex permittivity of the sample, and we show that reliable determination of the absorption coefficient from diffuse reflectance measurements therefore requires knowledge of the samples refractive index. This work demonstrates the applicability of diffuse reflectance measurements, using a THz frequency quantum cascade laser, to the high-resolution spectroscopic analysis of bulk powdered samples at THz frequencies.

Life in extreme environments: single molecule force spectroscopy as a tool to explore proteins from extremophilic organisms.

Extremophiles are organisms which survive and thrive in extreme environments. The proteins from extremophilic single-celled organisms have received considerable attention as they are structurally stable and functionally active under extreme physical and chemical conditions. In this short article, we provide an introduction to extremophiles, the structural adaptations of proteins from extremophilic organisms and the exploitation of these proteins in industrial applications. We provide a review of recent developments which have utilized single molecule force spectroscopy to mechanically manipulate proteins from extremophilic organisms and the information which has been gained about their stability, flexibility and underlying energy landscapes.

Single molecule force spectroscopy reveals the temperature-dependent robustness and malleability of a hyperthermophilic protein

The ability of thermophilic and hyperthermophilic proteins to maintain their native structure, yet be dynamic and flexible is a key determinant of their ability to function at the extremes of environmental temperatures found on Earth. An understanding of the design principles governing their material properties is important in the development of biomaterials which are able to withstand such extreme conditions. Single molecule force spectroscopy is used to characterise the mechanical flexibility of cold shock protein B from a hyperthermophilic organism, Thermotoga maritima, in the temperature range from 5–40 °C. We measure temperature-dependent changes in features of the unfolding energy landscape of this protein by studying the pulling speed dependence of the unfolding force with temperature in combination with Monte Carlo simulations. We find that the position of the transition state to unfolding shifts away from the native state with increased temperature, reflecting a reduction in the spring constant of the protein and an increase in the malleability of the structure. The mechanical robustness and malleability of this cold shock protein over the temperature range studied, provides an insight into the dynamical properties of hyperthermophilic proteins and lays the foundations for further studies using this highly structurally conserved protein family.

A Simple Transfer-Function-Based Approach for Estimating Material Parameters From Terahertz Time-Domain Data

A novel parametrically efficient approach to estimating the spectra of short transient signals is proposed and evaluated, with an application to estimating material properties, including complex refractive index and absorption coefficient, in the terahertz frequency range. This technique includes uncertainty analysis of the obtained spectral estimates, allowing rigorous statistical comparison between samples. In the proposed approach, a simple few-parameter continuous-time transfer function model explains over 99.9% of the measured signal. The problem, normally solved using poorly numerically defined Fourier transform deconvolution methods, is reformulated and cast as a time-domain dynamic-system estimation problem, thus providing a true time-domain spectroscopy tool.

Differential Effects of Hydrophobic Core Packing Residues for Thermodynamic and Mechanical Stability of a Hyperthermophilic Protein.

Proteins from organisms that have adapted to environmental extremes provide attractive systems to explore and determine the origins of protein stability. Improved hydrophobic core packing and decreased loop-length flexibility can increase the thermodynamic stability of proteins from hyperthermophilic organisms. However, their impact on protein mechanical stability is not known. Here, we use protein engineering, biophysical characterization, single-molecule force spectroscopy (SMFS), and molecular dynamics (MD) simulations to measure the effect of altering hydrophobic core packing on the stability of the cold shock protein TmCSP from the hyperthermophilic bacterium Thermotoga maritima. We make two variants of TmCSP in which a mutation is made to reduce the size of aliphatic groups from buried hydrophobic side chains. In the first, a mutation is introduced in a long loop (TmCSP L40A); in the other, the mutation is introduced on the C-terminal β-strand (TmCSP V62A). We use MD simulations to confirm that the mutant TmCSP L40A shows the most significant increase in loop flexibility, and mutant TmCSP V62A shows greater disruption to the core packing. We measure the thermodynamic stability (ΔGD-N) of the mutated proteins and show that there is a more significant reduction for TmCSP L40A (ΔΔG = 63%) than TmCSP V62A (ΔΔG = 47%), as might be expected on the basis of the relative reduction in the size of the side chain. By contrast, SMFS measures the mechanical stability (ΔG*) and shows a greater reduction for TmCSP V62A (ΔΔG* = 8.4%) than TmCSP L40A (ΔΔG* = 2.5%). While the impact on the mechanical stability is subtle, the results demonstrate the power of tuning noncovalent interactions to modulate both the thermodynamic and mechanical stability of a protein. Such understanding and control provide the opportunity to design proteins with optimized thermodynamic and mechanical properties.