New article in Nanoscale
04 October 2021Development of a universal conductive platform for anchoring photo- and electroactive proteins using organometallic terpyridine molecular wiresResolution enhancement in NMR spectra by deconvolution with compressed sensing reconstruction
Margot Jacquet, Miriam Izzo, Silvio Osella, Sylwia Kozdra, Paweł P. Michałowski, Dariusz Gołowicz, Krzysztof Kazimierczuk, Maciej T. Gorzkowski, Adam Lewera, Marian Teodorczyk, Bartosz Trzaskowski, Rafał Jurczakowski, Daniel T. Gryko ORCID and Joanna Kargul
The construction of an efficient conductive interface between electrodes and electroactive proteins is a major challenge in the biosensor and bioelectrochemistry fields to achieve the desired nanodevice performance. Concomitantly, metallo-organic terpyridine wires have been extensively studied for their great ability to mediate electron transfer over a long-range distance. In this study, we report a novel stepwise bottom-up approach for assembling bioelectrodes based on a genetically modified model electroactive protein, cytochrome c553 (cyt c553) and an organometallic terpyridine (TPY) molecular wire self-assembled monolayer (SAM). Efficient anchoring of the TPY derivative (TPY-PO(OH)2) onto the ITO surface was achieved by optimising solvent composition. Uniform surface coverage with the electroactive protein was achieved by binding the cyt c553 molecules via the C-terminal His6-tag to the modified TPY macromolecules containing Earth abundant metallic redox centres. Photoelectrochemical characterisation demonstrates the crucial importance of the metal redox centre for the determination of the desired electron transfer properties between cyt and the ITO electrode. Even without the cyt protein, the ITO-TPY nanosystem reported here generates photocurrents whose densities are 2-fold higher that those reported earlier for ITO electrodes functionalised with the photoactive proteins such as photosystem I in the presence of an external mediator, and 30-fold higher than that of the pristine ITO. The universal chemical platform for anchoring and nanostructuring of (photo)electroactive proteins reported in this study provides a major advancement for the construction of efficient (bio)molecular systems requiring a high degree of precise supramolecular organisation as well as efficient charge transfer between (photo)redox-active molecular components and various types of electrode materials.
Special Issue of Magnetic Resonance in Chemistry
“Applications of Alternative Sampling Methods” guest-edited by Krzysztof Kazimierczuk
New Article in Magnetic Resonance in Chemistry
09 November 2020Benefits of time‐resolved nonuniform sampling in reaction monitoring: The case of aza‐Michael addition of benzylamine and acrylamide
Dariusz Gołowicz, Magdalena Kaźmierczak and Krzysztof Kazimierczuk*
Time‐resolved nonuniform sampling (TR‐NUS) applied to reaction monitoring eliminates 2D spectral line disturbances resulting from varying peak intensities and positions. This allows, in turn, avoiding wrong conclusions about kinetics. These considerations are exemplified with a seemingly simple aza‐Michael reaction of benzylamine and acrylamide. Surprisingly, the product identification is possible only using 2D spectra, although credible monitoring requires TR‐NUS.
New Article in Analyst
07 September 2020Enhancing benchtop NMR spectroscopy by means of sample shifting
Benchtop NMR spectrometers have become widely available over the last decade. They are now used successfully in various branches of chemistry. Their popularity continues to grow due to their low price and almost zero running costs. However, benchtop spectrometers perform much less effectively than the high-field spectrometers used in NMR labs for several decades. In this article we present a solution for boosting the sensitivity of benchtop NMR spectrometers in a multi-scan experiment and improving its capabilities in quantitative measurement. Our solution involves the synchronized shifting of a sample to preserve its high nuclear polarization during the measurement. We performed several experiments using different samples to confirm this improved performance: an1H NMR experiment for 4,4-Dimethoxy-2-butanone, and13C NMR experiments for benzyl salicylate,liquid pharmaceutical product Acerin (skin solution), and a mixture of m-Anisaldehyde and (R)-(+)-Limonene.
New Article in J. of Biomolecular NMR
A novel high-dimensional NMR experiment for resolving protein backbone dihedral angle ambiguities
Clemens Kauffmann, Krzysztof Kazimierczuk, Thomas C. Schwarz, Robert Konrat & Anna Zawadzka-Kazimierczuk
Intrinsically disordered proteins (IDPs) are challenging established structural biology perception and urge a reassessment of the conventional understanding of the subtle interplay between protein structure and dynamics. Due to their importance in eukaryotic life and central role in protein interaction networks, IDP research is a fascinating and highly relevant research area in which NMR spectroscopy is destined to be a key player. The flexible nature of IDPs, as a result of the sampling of a vast conformational space, however, poses a tremendous scientific challenge, both technically and theoretically. Pronounced signal averaging results in narrow signal dispersion and requires higher dimensionality NMR techniques. Moreover, a fundamental problem in the structural characterization of IDPs is the definition of the conformational ensemble sampled by the polypeptide chain in solution, where often the interpretation relies on the concept of ‘residual structure’ or ‘conformational preference’. An important source of structural information is information-rich NMR experiments that probe protein backbone dihedral angles in a unique manner. Cross-correlated relaxation experiments have proven to fulfil this task as they provide unique information about protein backbones, particularly in IDPs. Here we present a novel cross-correlation experiment that utilizes non-uniform sampling detection schemes to resolve protein backbone dihedral ambiguities in IDPs. The sensitivity of this novel technique is illustrated with an application to the prototypical IDP 𝛼α-Synculein for which unexpected deviations from random-coil-like behaviour could be observed.
New Article in PLOS
Restriction of S-adenosylmethionine conformational freedom by knotted protein binding sites
Agata P. Perlinska, Adam Stasiulewicz, Ewa K. Nawrocka, Krzysztof Kazimierczuk, Piotr Setny, Joanna I. Sulkowska
S-adenosylmethionine (SAM) is one of the most important enzyme substrates. It is vital for the function of various proteins, including large group of methyltransferases (MTs). Intriguingly, some bacterial and eukaryotic MTs, while catalysing the same reaction, possess significantly different topologies, with the former being a knotted one. Here, we conducted a comprehensive analysis of SAM conformational space and factors that affect its vastness. We investigated SAM in two forms: free in water (via NMR studies and explicit solvent simulations) and bound to proteins (based on all data available in the PDB and on all-atom molecular dynamics simulations in water). We identified structural descriptors—angles which show the major differences in SAM conformation between unknotted and knotted methyltransferases. Moreover, we report that this is caused mainly by a characteristic for knotted MTs compact binding site formed by the knot and the presence of adenine-binding loop. Additionally, we elucidate conformational restrictions imposed on SAM molecules by other protein groups in comparison to conformational space in water.
