Apart from biological functions, peptides are of uttermost importance as models for un- folded, denatured or disordered state of the proteins. Similarly, miniproteins such as Trp-cage have proven their role as simple models of both experimental and theoretical studies of protein folding. Molecular dynamics and computer simulations can provide an unique insight on processes at atomic level. However, simulations of peptides and minipro- teins face two cardinal problems-inaccuracy of force fields and inadequate conformation sampling. Both principal issues were tackled in this theses. Firstly, the differences in several force field for peptides and proteins were questioned. We demonstrated the inability of the used force fields to predict consistently intrinsic conformational preferences of individual amino acids in the form of dipeptides and the source of the discrepancies was traced. In order to shed light on the nature of conformational ensembles under various denatur- ing conditions, we studied host-guest AAXAA peptides. The simulations revealed that thermal and chemical denaturation by urea produces qualitatively different ensembles and shift propensities of individual amino acids to particular conformers. The problem of insufficient conformation sampling was dealt by introducing gyration- and...

DNA is the fundamental molecule in all domains of life, its role in heredity is well established. Although the famous double helical complementary form is indispensable for replication mechanism DNA can occupy wide range of conformations. In the past studies performed in the laboratory, DNA oligomers related to single stranded bacterial Repetitive Extragenic Palindromic (REP) showed spectral behavior suggesting complex equilibria including double helical, hairpin, and tetraplex conformations. The studies presented in this thesis extended the scope of analyzed sequences and employed circular dichroism spectroscopy and X-ray crystallography. We report spectral data and X-ray structures of three successfully crystalized oligonucleotides. All three structures acquire double helical architecture with two consecutive T- T mismatches in the center. To improve the convergence of the refinement process of the crystal structures we used novel dinucleotide conformational classes, NtC classes. The NtC class classification was also used to analyze geometries of selected non-canonical base pairs in all DNA crystal structures in the Protein Data Bank. We measured the fit between geometries of the dinucleotides involved in the non-canonical base pairing and the NtC classes and correlated this fit to the electron...

Bordetella pertussis is a strictly human pathogen and causative agent of infectious respiratory disease called whooping cough. In order to establish successful infection and colonization of the host, B. pertussis uses a broad spectrum of virulence factors such as adhesins (filamentous hemagglutinin, pertactin, and fimbriae) and toxins (adenylate cyclase and pertussis toxins). In addition, the type 3 secretion system (T3SS) was also found in the genus Bordetella. In connection to our previous characterisation of B. pertussis strain lacking the gene encoding RNA chaperone Hfq (Δhfq), which proved that Hfq is required for T3SS functionality, the recombinant T3SS proteins BopB, BopD, BopC and BopN were purified to homogeneity. Next, the specific antibodies were obtained using purified recombinant proteins in order to study the production of the T3SS components in B. pertussis. Using refined anti- BopC antibodies it was for the first time shown that laboratory-adapted B. pertussis strain secretes BopC protein into medium. The recombinant translocators BopB and BopD were also used to examine their pore-forming activity using planar black lipid membranes. Based on the characterisation of hfq deletion mutant, having impaired production of membrane proteins when compared to the wild type, mass spectrometry...

Humic acids are part of the natural organic matter occurring all around us. The aim of this thesis is to study the molecular organization, conformation of humic acids in aqueous solutions, that always raise a number of questions. For a long time, the scientists all over the world argue, if humic acid are polymers, micelles or supramolecules. Over time, thanks to new technologies, their opinions are moving away from a polymer model and tend rather to supramolecular arrangement of humic acids. Studying humic complex systems is not easy, because they are polydisperse and heterogeneous, which significantly complicates any characterization. Moreover, its molecular organization is affected by many factors such as e.g. pH, ionic strength and etc. For the study of the conformation of humic acids, concentration series of IHSS (International humic substances asociation) Leonardite humic acids stamdards in four different mediums were prepared: 0,1 M NaOH, humic acids in water with pH modified to 12 (basic medium), 0,1 M NaOH + 0,1 M HCl, 0,1 M NaCl (neutral environment). Furthermore it has been performed the fractionation of humic acids for the purpose of simplifying the complicated structure. Prepared concentration series were characterized with several analytical methods such as ultraviolet and visible spectroscopy, dynamic light scattering, electrophoretic light scattering, microrheology, gel permeation chromatography, potentiometric pH determination and direct conductometry. Diploma thesis is built on the previous bachelor thesis in which different sample of humic acids was studied. The measured results indicate that the studied systems show supramolecular behavior and in some cases are subject to aggregation into larger units (micelles).

Bacterial protein WrbA from E. coli is the founding member of a new family of FMN-dependent NAD(P)H oxidoreductases, forming a functional and structural bridge between bacterial flavodoxin and certain mammalian NAD(P)H:quinone oxidoreductase. For these reasons, protein WrbA is recently intensively studied using various analytical and computing methods. Protein WrbA participates in the protection of cells against oxidative stress, but precise function of the protein WrbA in vivo is still unknown. Protein WrbA forms multimers in solutions. In μM concentrations and at low temperature (4 řC) the protein is in the form of a dimer, with increasing temperature becomes tetrameric. Available three-dimensional crystal structure contains the information about the tetrameric form of the protein, the dimeric form has not been structurally characterized. This thesis was focused on the study of the dynamic behavior of protein WrbA in solution using methods of hydrogen-deuterium exchange and chemical cross-linking followed by mass spectrometric analysis with high resolution (FT-ICR). Behavior of the protein was monitored according to the presence of cofactor FMN. Effect of temperature and protein concentration was also studied. Hydrogen-deuterium exchange provided information about solvent accessibility and...

This diploma thesis deals with the conformation and molecular organization of humic acids in aqueous solutions. Humic substances have heterogeneous and polydisperse nature, therefore their secondary chemical structure has not yet been defined properly, neither has their conformational arrangement. The conformation of humic substances in the solutions is mainly stabilized by weak disperse forces, such as Van der Waals, -, CH- interactions and hydrogen bonds. Humic substances in the solutions tend to form aggregates that are held together mostly by the intermolecular hydrophobic interactions. Concentration series of humic acids were prepared in three different mediums of constant ionic strength: NaOH and NaCl (prepared either by neutralization NaOH by HCl or direct dilution by solution of NaCl). Several analytical methods have been used to determine conformation and molecular organization of humic acids: potentiometric determination of pH, direct conductometry, ultraviolet and visible spectroscopy, density measurement, dynamic light scattering, laser Doppler velocimetry and high resolution ultrasound spectrometry. It was confirmed that the conformation and molecular organization of humic acids in aqueous solutions depend not only on pH of medium, but they also depend on chemical nature and concentration with the same pH. Results showed that hydrodynamic diameter of particles is significantly increasing in NaCl medium prepared by neutralization NaOH by HCl, particularly at low concentrations, which corresponds to higher values of zeta potential obtained. Concentration dependencies of ultrasonic velocity and compressibility also indicate the changes in conformation and molecular organization corresponding with results from other methods used.