Nikola Paul Chmel

Background fluorescence remains the biggest challenge in Raman spectroscopy because of the consequent curvature of the baseline and the degradation of the signal-to-noise ratio of the Raman signal. While the concentrations of the fluorophore impurities are usually too low to be detected by other analytical methods, they are often sufficient to prevent Raman data collection. Among the different existing methods to remove the fluorescence signal, photobleaching remains the most popular due to its simplicity. However, using the spectrometer laser to photobleach is far from optimal. Most commercially available instruments have little or no choice of wavelength, and their output powers are in many cases not suitable for highly fluorescent samples such as those from biological systems (e.g., proteins). In this article, we assess practical aspects of photobleaching such as the apparent reversibility of the process and the effect of convection currents due to what we speculate to be temperature gradients across the bulk of the solution. We also introduce an affordable custom made external photobleaching unit with a choice of excitation wavelength and demonstrate its viability with a highly fluorescent bovine serum albumin protein solution, which had proved most challenging for Raman spectroscopy as it contained ∼10% w/w impurities.

Background: Proteins are biomolecules that consist of sequences of amino acids (primary structure) which can further interact and cause the backbone to fold into more complex arrangements (secondary and tertiary structures). Any chemical alterations of the molecules after the translation of the messenger RNA code into a protein primary sequence are known as posttranslational modifications (PTMs). PTMs may affect the protein’s functionality; thus it is necessary to identify them. PTMs of particular interest to the pharmaceutical industry include deamidation, oxidation, deglycosylation and isomerization, which may occur due to environmental stressors. However, they have proved challenging to identify quickly. Electronic and vibrational spectroscopies have proved valuable tools for studying higher-order structure and stability of proteins. Materials & Methods: In this work, circular dichroism (CD), infrared absorbance (IR) and Raman spectroscopies were applied to characterize antibody (mAb NIP 228) PTMs as a result of different stressors. Mass spectrometry was used to confirm the identity of modifications including the targeted ones. Room temperature CD showed that the secondary structure was the same after all treatments, and temperature-controlled CD showed how protein stability was affected by modifications. Both Raman and IR analysis detected small differences between the reference and deglycosylated proteins, and clearly indicated the presence of other PTMs. Conclusion: This work required some novel computational approaches to pre-process Raman and IR spectra and a review of the band assignments for proteins existing in the literature.

We investigate the preparation of polysiloxane-based networks under solvent-free, ambient conditions using the Lewis acid catalysed Piers-Rubinsztajn (PR) reaction of hydride-terminated siloxanes with various tetrafunctional alkoxysilanes (tetraethoxysilane, tetrapropoxysilane, tetra-n-buxoxysilane, tetra-s-butoxysilane, tetra-s-butoxysilane, and tetrakis(2-ethylbutoxy)silane) as crosslinkers. We explore the effects of polysiloxane chain length and crosslinker alkyl group on the rheological performance of the elastomers. By analysing the reaction progress by grazing angle Fourier-transform infrared spectroscopy (FTIR) and determining the rheological properties of the resulting materials, we show that the use of linear or branched alkoxysilanes strongly influences the morphology and properties of these network polymers. We have shown the PR process can be tailored to reliably produce homogeneous, polysiloxane network materials. This work provides information on the relative rates of network formation under ambient conditions with an emphasis on the impact of crosslinker alkyl chain length. Our results show that electronics and sterics both play critical roles in influencing the rate of the curing reaction. Crucially, we newly demonstrate the benefit of a having tertiary carbon α to the SiO reaction centre, as is the case for the tetra-s-butoxysilane crosslinker, for delivering exceptionally rapid network cure and a concomitant enhancement in storage modulus of the resultant materials.

Infrared (IR) spectroscopy is increasingly being used to probe the secondary structure of proteins, especially for high-concentration samples and biopharmaceuticals in complex formulation vehicles. However, the small path lengths required for aqueous protein transmission experiments, due to high water absorbance in the amide I region of the spectrum, means that the path length is not accurately known, so only the shape of the band is ever considered. This throws away a dimension of information. Attenuated total reflectance (ATR) IR spectroscopy is much easier to implement than transmission IR spectroscopy and, for a given instrument and sample, gives reproducible spectra. However, the ATR-absorbance spectrum varies with sample concentration and instrument configuration, and its wavenumber dependence differs significantly from that observed in transmission spectroscopy. In this paper, we determine, for the first time, how to transform water and aqueous protein ATR spectra into the corresponding transmission spectra with appropriate spectral shapes and intensities. The approach is illustrated by application to water, concanavalin A, haemoglobin and lysozyme. The transformation is only as good as the available water refractive index data. A hybrid of literature data provides the best results. The transformation also allows the angle of incidence of an ATR crystal to be determined. This opens the way to using both spectral shape and spectra intensity for protein structure fitting.

A bipyridyl reagent containing a strained alkyne 7, reacts with benzyl azide to give a significantly more flexible product 10 and an increase in fluorescence is observed. Upon addition of Zn(ii), the fluorescence is enhanced further.

EB proteins track the ends of growing microtubules and regulate microtubule dynamics both directly and by acting as the hub of the tip-tracking network. Mammalian cells express cell type-specific combinations of three EB proteins with different cellular roles. Here, we reconstitute EB1, EB2 and EB3 tip tracking in vitro. We find that all three EBs show rapid exchange at the microtubule tip and that their signal correlates to the microtubule assembly rate. However, the three signals differ in their maxima and position from the microtubule tip. Using microtubules built with nucleotide analogues and site-directed mutagenesis, we show that EB2 prefers binding to microtubule lattices containing a 1:1 mixture of different nucleotides and its distinct binding specificity is conferred by amino acid substitutions at the right-hand-side interface of the EB microtubule-binding domain with tubulin. Our data are consistent with the model that all three EB paralogues sense the nucleotide state of both β-tubulins flanking their binding site. Their different profile of preferred binding sites contributes to occupying spatially distinct domains at the temporally evolving microtubule tip structure.

We demonstrate that by measuring the fluorescence emission data during a linear dichroism measurement, one obtains additional information on the exited state properties and interactions of oriented molecules in the solid state.

Attenuated total reflectance (ATR) infrared absorbance spectroscopy of proteins in aqueous solution is much easier to perform than transmission spectroscopy, where short path‐length cells need to be assembled reproducibly. However, the shape of the resulting ATR infrared spectrum varies with the refractive index of the sample and the instrument configuration. Refractive index in turn depends on the absorbance of the sample. In this work, it is shown that a room temperature triglycine sulfate detector and a ZnSe ATR unit can be used to collect reproducible spectra of proteins. A simple method for transforming the protein ATR spectrum into the shape of the transmission spectrum is also given, which proceeds by approximating a Kramers‐Krönig–determined refractive index of water as a sum of four linear components across the amide I and II regions. The light intensity at the crystal surface (with 45° incidence) and its rate of decay away from the surface is determined as a function of the wave number–dependent refractive index as well as the decay of the evanescent wave from the surface. The result is a single correction factor at each wave number. The spectra were normalized to a maximum of 1 between 1600 cm⁻¹ and 1700 cm⁻¹ and a self‐organizing map secondary structure fitting algorithm, SOMSpec, applied using the BioTools reference set. The resulting secondary structure estimates are encouraging for the future of ATR spectroscopy for biopharmaceutical characterization and quality control applications.

Fluorescence detection typically enhances sensitivity and selectivity for fluorescent analytes. The potential for combining fluorescence detection with flow orientation of the sample in the normal configuration of linear dichroism experiments is explored in this work by measuring the fluorescence emitted from flow‐orientated DNA‐bound ligands and M13 bacteriophage. Data for ethidium bromide, Hoechst 33258, and 4,6‐diamidino‐2‐phenyindole are presented. The theoretical basis of the technique is also presented for instruments running in both the fixed direct‐current mode, which is the normal operation mode of circular dichroism spectropolarimeters, and also in fixed high‐tension voltage mode. The role of the stray light reaching the detector that results in a spectral shape in fixed direct current mode that resembles the shape of a linear dichroism spectrum, rather than the expected reduced linear dichroism, is also explored.