Biomedical Spectroscopy and Imaging - Volume Preprint, issue Preprint
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Biomedical Spectroscopy and Imaging (BSI) is a multidisciplinary journal devoted to the timely publication of basic and applied research that uses spectroscopic and imaging techniques in different areas of life science including biology, biochemistry, biotechnology, bionanotechnology, environmental science, food science, pharmaceutical science, physiology and medicine. Scientists are encouraged to submit their work for publication in the form of original articles, brief communications, rapid communications, reviews and mini-reviews.
The journal is dedicated to providing a single forum for experts in spectroscopy and imaging as applied to biomedical problems, and also for life scientists who use these powerful methods for advancing their research work. BSI aims to promote communication, understanding and synergy across the diverse disciplines that rely on spectroscopy and imaging. It also encourages the submission of articles describing development of new devices and technologies, based on spectroscopy and imaging methods, for application in diverse areas including medicine, biomedical science, biomaterials science, environmental science, pharmaceutical science, proteomics, genomics, metabolomics, microbiology, biotechnology, genetic engineering, nanotechnology, etc.
Abstract: This article reviews work presented at the ESCBM 2017 on using two-dimensional infrared (2D IR) spectroscopy to probe ion binding configurations in the potassium ion channel KcsA. We discuss two studies in which we use 2D IR spectroscopy in conjunction with protein semisynthesis and molecular dynamics (MD) simulations to test against two competing models of ion permeation and to investigate how changes in the conformation of the intracellular gate affect the structure and ion affinities of the selectivity filter. In our initial study, the 2D IR spectrum of an isotopically labeled KcsA filter reveals two spectral features that correspond to…different structures and ion binding configurations. MD simulations help us link the experiments to atomistic structures, allowing us to determine the prevailing mechanism of ion conduction. In a follow-up study, we probe how ion occupancies in the filter change with the opening and closing of the intracellular gate. We experimentally modulate the conformation of KcsA using different sample conditions, and are able to show that the ion occupancies in the filter change with the state of the gate, revealing complexities in the conformational landscape of the potassium ion channel. Our work shows the potential of these techniques in addressing the fundamental biophysical questions in membrane protein structure and function.
Keywords: 2D IR spectroscopy, potassium ion channels, ion permeation, protein semisynthesis
Abstract: Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy is a surface-sensitive and label-free technique, which is applied to obtain dynamic structural information of biomolecules. The study of proteins by ATR-FTIR spectroscopy can be impeded by their tendency to adsorb to solid surfaces. Furthermore, the adsorption process of proteins is often accompanied with conformational changes, which can interfere with the intended structural analysis. We efficiently modified a silicon ATR crystal surface with polyethylene glycol and thereby create a protein-repellent surface. To achieve a high sensitivity, which enables the study of small conformational changes of biomolecules, we combine surface passivation with specific immobilization.…This is accomplished via the biotin-streptavidin interaction, which is one of the strongest known non-covalent protein-ligand interactions. As a proof of concept we present the specific immobilization of DNA. The modified surface is stable against elevated temperatures and 8 M urea and can therefore be used to study a wide range of biochemical systems and reactions. The surface chemistry is simple and performed under mild conditions, which leads to a high applicability of the presented approach.
Keywords: ATR-FTIR spectroscopy, biomolecules, protein adsorption, surface passivation, specific immobilization
Abstract: Solid supported lipid bilayers (SSLB) play an important role as biomimetic membranes to study protein-membrane interactions. We investigated the orientation of lipids in SSLBs at different temperatures and over time. Especially the stability of the lipid bilayer and structural changes upon lipid phase transition were analyzed by polarized ATR-FTIR spectroscopy and with SSLBs of different lipid compositions. The integrity of a lipid bilayer consisting of POPC or a 1:1 mixture of POPC and POPG is conserved over a wide temperature range and over several hours. Furthermore, we were able to monitor changes in the orientation of the lipid alkyl chains…upon lipid phase transition for DMPC and DSPC. This study shows that the combination of solid supported lipid bilayers and polarized ATR-FTIR spectroscopy is very powerful to characterize lipid membranes under different environmental conditions. The sensitivity of this technique will be exploited in future studies to analyze the effect of protein-membrane interaction on lipid orientation.