A Delocalized Proton-Binding Site within a Membrane Protein

AbstractThe role of protein-bound water molecules in protein function and catalysis is an emerging topic. Here, we studied the solvation of an excess proton by protein-bound water molecules and the contribution of the surrounding amino acid residues at the proton release site of the membrane protein bacteriorhodopsin. It hosts an excess proton within a protein-bound water cluster, which is hydrogen bonded to several surrounding amino acids. Indicative of delocalization is a broad continuum absorbance experimentally observed by time-resolved Fourier transform infrared spectroscopy. In combination with site-directed mutagenesis, the involvement of several amino acids (especially Glu-194 and Glu-204) in the delocalization was elaborated. Details regarding the contributions of the glutamates and water molecules to the delocalization mode in biomolecular simulations are controversial. We carried out quantum mechanics/molecular mechanics (QM/MM) self-consistent charge density functional tight-binding simulations for all amino acids that have been experimentally shown to be involved in solvation of the excess proton, and systematically investigated the influence of the quantum box size. We compared calculated theoretical infrared spectra with experimental ones as a measure for the correct description of excess proton delocalization. A continuum absorbance can only be observed for small quantum boxes containing few amino acids and/or water molecules. Larger quantum boxes, including all experimentally shown involved amino acids, resulted in narrow absorbance bands, indicating protonation of a single binding site in contradiction to experimental results. We conclude that small quantum boxes seem to reproduce representative extreme cases of proton delocalization modes: proton delocalization only on water molecules or only between Glu-194 and Glu-204. Extending the experimental spectral region to lower wave numbers, a water-delocalized proton reproduces the observed continuum absorbance better than a glutamate-shared delocalized proton. However, a full agreement between QM simulations and experimental results on the delocalized excess proton will require a larger quantum box as well as more sophisticated QM/MM methods

Shape Decomposition Algorithms for Laser Capture Microdissection

In the context of biomarker discovery and molecular characterization of diseases, laser capture microdissection is a highly effective approach to extract disease-specific regions from complex, heterogeneous tissue samples. These regions have to be decomposed into feasible fragments as they have to satisfy certain constraints in size and morphology for the extraction to be successful. We model this problem of constrained shape decomposition as the computation of optimal feasible decompositions of simple polygons. We use a skeleton-based approach and present an algorithmic framework that allows the implementation of various feasibility criteria as well as optimization goals. Motivated by our application, we consider different constraints and examine the resulting fragmentations. Furthermore, we apply our method to lung tissue samples and show its advantages in comparison to a heuristic decomposition approach

Grayscale representation of infrared microscopy images by Extended Multiplicative Signal Correction for registration with histological images

Fourier-transform infrared (FTIR) microspectroscopy is rounding the corner to become a label-free routine method for cancer diagnosis. In order to build infrared-spectral based classifiers, infrared images need to be registered with Hematoxylin and Eosin (H&E) stained histological images. While FTIR images have a deep spectral domain with thousands of channels carrying chemical and scatter information, the H&E images have only three color channels for each pixel and carry mainly morphological information. Therefore, image representations of infrared images are needed that match the morphological information in H&E images. In this paper, we propose a novel approach for representation of FTIR images based on extended multiplicative signal correction highlighting morphological features that showed to correlate well with morphological information in H&E images. Based on the obtained representations, we developed a strategy for global-to-local image registration for FTIR images and H&E stained histological images of parallel tissue sections.publishedVersio

Proton uptake mechanism of bacteriorhodopsin as determined by time-resolved stroboscopic-FTIR-spectroscopy

Bacteriorhodopsin's proton uptake reaction mechanism in the M to BR reaction pathway was investigated by time-resolved FTIR spectroscopy under physiological conditions (293 K, pH 6.5, 1 M KCl). The time resolution of a conventional fast-scan FTIR spectrometer was improved from 10 ms to 100 μs, using the stroboscopic FTIR technique. Simultaneously, absorbance changes at 11 wavelengths in the visible between 410 and 680 nm were recorded. Global fit analysis with sums of exponentials of both the infrared and visible absorbance changes yields four apparent rate constants, k(7) = 0.3 ms, k(4) = 2.3 ms, k(3) = 6.9 ms, k(6) = 30 ms, for the M to BR reaction pathway. Although the rise of the N and O intermediates is dominated by the same apparent rate constant (k(4)), protein reactions can be attributed to either the N or the O intermediate by comparison of data sets taken at 273 and 293 K. Conceptionally, the Schiff base has to be oriented in its deprotonated state from the proton donor (asp 85) to the proton acceptor (asp 96) in the M(1) to M(2) transition. However, experimentally two different M intermediates are not resolved, and M(2) and N are merged. From the results the following conclusions are drawn: (a) the main structural change of the protein backbone, indicated by amide I, amide II difference bands, takes place in the M to N (conceptionally M(2)) transition. This reaction is proposed to be involved in the “reset switch” of the pump, (b) In the M to N (conceptionally M(2)) transition, most likely, asp-85's carbonyl frequency shifts from 1,762 to 1,753 cm(-1) and persists in O. Protonation of asp-85 explains the red-shift of the absorbance maximum in O. (c) The catalytic proton uptake binding site asp-96 is deprotonated in the M to N transition and is reprotonated in O