|
| |
|
| |
|
| |
|
|
Study of protein structure and dynamics by means of optical spectroscopy
Pazderka, Tomáš ; Kopecký, Vladimír (advisor) ; Kaminský, Jakub (referee) ; Setnička, Vladimír (referee)
Title: Study of protein structure and dynamics by means of optical spectroscopy Author: Tomáš Pazderka Institute: Institute of Physics of Charles University Supervisor: RNDr. Vladimír Kopecký, Ph.D., Institute of Physics of Charles University Abstract: The aim of this thesis is to improve understanding of protein structure and dynamics and extend experimental setup and data processing for such stud- ies. We focus on the extension of experimental feasability of vibrational optical activity (VOA). We have demonstrated a usability of intensity calibration in the field of Raman optical activity. Advantages for measurements on multiple instru- ments and/or using different configurations have been shown. A new instrumental setup has been developed for microsampling measurements of vibrational circular dichroism spectra with a spatial resolution of 1 mm. Using this technique, spatial inhomogeneities in a sample of protein fibrils have been observed. Model com- pounds for amide nonplanarity have been investigated utilizing several methods of optical spectroscopy and key spectral features for determination of amide non- planarity and the absolute configuration have been identified. A comprehensive set of Raman spectra of proteinogenic amino acids has been measured. Sample concentration dependencies and consequent...
|
|
|
Never Born Proteins: Occurence and characterization of secondary structure motifs
Treťjačenko, Vjačeslav ; Hlouchová, Klára (advisor) ; Kopecký, Vladimír (referee)
An experimental study on randomly generated protein sequences can provide important insights into the origin and mechanism of secondary structure formation and protein folding. In this study we bring biophysical characterization of five protein sequences selected from the in silico generated library of random chains. The sequences were selected on the basis of bioinformatic analysis in order to find the candidates with the maximum potential to possess secondary structure. This study shows that the random polypeptide sequences form stable secondary structures and in some show the signs of tertiary structure, such as hydrophobic core formation and distinctive oligomerization pattern. While the work presented in this thesis is work in progress on a larger study, the data already demonstrate that unevolved protein sequence space provides a lot of potential for secondary and tertiary structure formation that awaits its characterization. Powered by TCPDF (www.tcpdf.org)
|
|
|
Protein dynamics studied by means of optical spectroscopy
Pazderka, Tomáš ; Kopecký, Vladimír (advisor) ; Maloň, Petr (referee)
In this diploma thesis a software for Two-dimensional correlation spectroscopy (2DCoS) was programmed and tested. Possible spectral phenomena in 2DCoS analysis of vibrational circular dichroism were tested on model cases. Thermal and time dependent series of Raman and Raman optical aktivity spectra of lysozyme protein were measured for practical applications of 2DCoS. Fibrillation and denaturation process of lysozyme was analyzed by means of 2DCoS and Factor analysis (FA). Proteins NKR_P1A and NKR_P1C were also studied and their thermal dynamics was monitored by means of Fourier transformed infrared spectroscopy and Raman spectroscopy. Dynamical behaviour of all studied proteins was characterized using 2DCoS.
|
|
|
Characterization of proteins of 2'-5' oligoadenylate pathway by means of vibrational spectroscopy
Víšová, Ivana ; Kopecký, Vladimír (advisor) ; Bednárová, Lucie (referee)
The work concerns to structural characterization of two important proteins of 2'-5' oligoadenylate pathway participating in an immune response of organism to a viral infection. Studied proteins were ankyrin domain of mouse RNase L, the C-terminal part of human phosphodiesterase 12 and the complete human phosphodiesterase 12. The proteins were characterized by Raman spectroscopy, infrared spectroscopy, electronic circular dichroism, dynamic light scattering and in addition by two non-spectroscopic methods- differential calorimetry and electrophoresis. For each protein the secondary structures, thermal stability, weight of oligomers and generally a basic characterization by above mentioned methods were provided.
|
|
|
Atomic force microscopy in the region of biomacromolecules
Vančura, Martin ; Kopecký, Vladimír (advisor) ; Bednárová, Lucie (referee)
Atomic force microscopy (AFM) enables sample imaging at the micro and nanoscale. Recently, the method is applied to investigate biomacromolecules. Here, we describe the basic principles of AFM with a special emphasis for bioapplications. We tested experimental abilities of Alpha 300 - the Raman microscope with AFM/SNOM accessory from WITec company. The ability of AFM to study objects of cellular dimensions was demonstrated on erythrocytes and green algae Desmodesmus quadricauda. We were able to observe growing of lysozyme protein fibrils on day scale - from dimensions of seeds (~3 nm height) up to fibrils itself (3-10 nm height and 100 nm up to micrometers length). Subsequently, we observed separate protein molecules of thyroglobulin (~6 nm) and also γ-globulin (~3 nm). It seems plausible to image objects up to 2 nm dimensions by the given device with respect to the signal/noise ratio.
|
|
|
Study of nucleic acids by means of drop coating deposition Raman microspectroscopy (DCDR)
Souček, Pavel ; Kopecký, Vladimír (advisor) ; Mojzeš, Peter (referee)
The work was focused on possibilities of the new technique of nonresonance Raman spectroscopy - drop coating deposition Raman (DCDR) spectroscopy upon study of nucleic acids. DCDR spectroscopy is based on deposition of a small droplet of the studied sample on a hydrophobic surface, where after evaporation of the solvent, ring of the studied material in glass phase is formed. That way the material is concentrated and upon measurements by means of Raman microspectrometry the increase of signal of several orders of magnitude is observed with respect to the standard technique of sample measurements in solvent. In this work we studied behavior of DNA 12-mer and DNA with 3000 base pairs. Our research showed that DCDR spectra of DNA dissolved in deionized water can be measured up to concentration of 30 M per base. The DCDR spectra are similar to those measured from solvents. Nevertheless, the deposited sample never forms a ring. It was shown that the size of nucleic acids didn't play dominant role upon ring formation but their charge which lead to repulsion of the molecules. After adding of sodium ions and subsequently also magnesium ions the ring formation was observed. Factor analysis of spectral maps demonstrated that formed rings are not fully homogenous and that upon their origin condensation of DNA could...
|
|
|
Raman spectroscopy as the tool for Alzheimer's disease diagnostics
Tesař, Adam ; Kopecký, Vladimír (advisor) ; Jelínek, Otakar (referee)
Alzheimer disease (AD) is the most frequent dementia. The prevalence is approximately 10% in 65 years old people. The current treatment is only progression protective, therefore it is crucial to find a new diagnostic approach for diagnosing AD in early stage. We analysed a set of 55 patients by the drop coating deposition Raman spectroscopy with the goal to verify previously published high sensitivity of the AD spectroscopic diagnosis in cerebral spinal fluid (CSF) and to find a new diagnostic method for blood serum (BS). We optimized measurement conditions for BS. The results were evaluated by the cluster analysis and the principal component analysis. The small set of samples exhibited high sensitivity in both CSF and BS but that distinctly decreased in the whole set. The results for CSF were affected by the choice of the analysed spectral interval. The best for AD diagnose was the interval containing peaks at 980, 1080 and 1249 cm-1.The results for BS have been the most sensitive in the whole spectral range. They have low sensitivity but high specificity for AD (92%). The usage of neural networks has conversely high sensitivity and low specificity in both sets of samples of BS and CSF. Powered by TCPDF (www.tcpdf.org)
|
guest ::
RSS feed.