З вірою в ренесанс Просвітництва, духовності, справедливості (Олександрові Миколайовичу Костенку – 60!)

У жовтні 2009 р. виповнилося 60 років докторові юридичних наук, професору, завідувачеві відділу кримінального права, кримінології та судоустрою Інституту держави і права ім. В.М. Корецького Олександрові Миколайовичу Костенку, творча діяльність якого багато років пов’язана з «Вісником Національної академії наук України». Олександр Миколайович – відомий в Україні та за її межами юрист, кримінолог, філософ, автор понад 300 наукових і науково-публіцистичних праць, у тому числі 4 індивідуальних і 15 колективних монографій. Намагаючись (під враженням від творчості Ф.М. Достоєвського) проникнути у таємницю феномену зла (зокрема злочинності і беззаконня), він прийшов до висновку, що воно є проявом людської сваволі і для його наукового дослідження треба розробити новий методологічний інструментарій пізнання соціальних явищ. Тому О. М. Костенко розвиває натуралістичний світогляд, виклавши свою доктрину в низці статей і книг

Cys-Ph-TAHA: a lanthanide binding tag for RDC and PCS enhanced protein NMR

Here we present Cys-Ph-TAHA, a new nonadentate lanthanide tag for the paramagnetic labelling of proteins. The tag can be easily synthesized and is stereochemically homogenous over a wide range of temperatures, yielding NMR spectra with a single set of peaks. Bound to ubiquitin, it induced large residual dipolar couplings and pseudocontact shifts that could be measured easily and agreed very well with the protein structure. We show that Cys-Ph-TAHA can be used to label large proteins that are biochemically challenging such as the Lac repressor in a 90 kDa ternary complex with DNA and inducer

Interplay of Protein and DNA Structure Revealed in Simulations of the lac Operon

The E. coli Lac repressor is the classic textbook example of a protein that attaches to widely spaced sites along a genome and forces the intervening DNA into a loop. The short loops implicated in the regulation of the lac operon suggest the involvement of factors other than DNA and repressor in gene control. The molecular simulations presented here examine two likely structural contributions to the in-vivo looping of bacterial DNA: the distortions of the double helix introduced upon association of the highly abundant, nonspecific nucleoid protein HU and the large-scale deformations of the repressor detected in low-resolution experiments. The computations take account of the three-dimensional arrangements of nucleotides and amino acids found in crystal structures of DNA with the two proteins, the natural rest state and deformational properties of protein-free DNA, and the constraints on looping imposed by the conformation of the repressor and the orientation of bound DNA. The predicted looping propensities capture the complex, chain-length-dependent variation in repression efficacy extracted from gene expression studies and in vitro experiments and reveal unexpected chain-length-dependent variations in the uptake of HU, the deformation of repressor, and the folding of DNA. Both the opening of repressor and the presence of HU, at levels approximating those found in vivo, enhance the probability of loop formation. HU affects the global organization of the repressor and the opening of repressor influences the levels of HU binding to DNA. The length of the loop determines whether the DNA adopts antiparallel or parallel orientations on the repressor, whether the repressor is opened or closed, and how many HU molecules bind to the loop. The collective behavior of proteins and DNA is greater than the sum of the parts and hints of ways in which multiple proteins may coordinate the packaging and processing of genetic information. © 2013 Czapla et al

NMR studies of the allosteric effectors of the lac operon

The aim of this thesis is to characterize the regulatory mechanism of the Lac repressor which is the molecular switch of the lac operon. Lac repressor binds to its cognate DNA operator and inhibits transcription. When an inducer binds to the protein, it triggers a conformational change that releases the protein from the operator. Thus the DNA operator and inducer are two allosteric effectors of the Lac repressor. In the first part of this work the structures of a dimeric mutant of the Lac repressor DNA binding domain (‘headpiece’) complexed with the auxiliary operators O2 and O3 were determined by NMR spectroscopy. To understand the mechanism of repressor-operator recognition these structures were compared to the previously determined structures of headpiece bound to the main operator O1 and to non-specific DNA. Headpiece shows significant conformational plasticity to compensate for small changes in its cognate DNA sequences. The difference in the affinities of the Lac repressor for its natural operators can be well explained by the current structures. To gain insight into the allosteric link between inducer binding and DNA affinity, deuterated dimeric Lac repressor and isolated ‘core’ domain, containing only the inducer binding site, were produced. NMR chemical shift changes between various regulatory states of the intact Lac repressor dimer (free, protein-inducer complex, protein-DNA complex, and a ternary protein-DNA-inducer complex) could be monitored. When mapped on available X-ray structures, they show the allosteric connection between inducer binding and DNA affinity and the residues involved in this signaling path. Our findings show that the dimeric Lac repressor is in a dynamic equilibrium between two conformational states, which can be switched to either the operator bound and the inducer bound states by the addition of either inducer. This picture is gives strong molecular support for the classical model of allostery of the Lac repressor proposed by J. Monod, J. Wyman and J-P. Changeux. Furthermore data from ternary complex, revealed how inducer binding results in the disruption of the contacts between the inducer binding domain and DNA binding domains, thereby destabilizing the dimerization interface within the DNA binding domain. This causes the release of the Lac repressor from the operator DNA

Specificity and affinity of Lac repressor for the auxiliary operators O2 and O3 are explained by the structures of their protein–DNA complexes

The structures of a dimeric mutant of the Lac repressor DNA-binding domain complexed with the auxiliary operators O2 and O3 have been determined using NMR spectroscopy and compared to the structures of the previously determined Lac–O1 and Lac–nonoperator complexes. Structural analysis of the Lac–O1 and Lac–O2 complexes shows highly similar structures with very similar numbers of specific and nonspecific contacts, in agreement with similar affinities for these two operators. The left monomer of the Lac repressor in the Lac–O3 complex retains most of these specific contacts. However, in the right half-site of the O3 operator, there is a significant loss of protein–DNA contacts, explaining the low affinity of the Lac repressor for the O3 operator. The binding mode in the right half-site resembles that of the nonspecific complex. In contrast to the Lac–nonoperator DNA complex where no hinge helices are formed, the stability of the hinge helices in the weak Lac–O3 complex is the same as in the Lac–O1 and Lac–O2 complexes, as judged from the results of hydrogen/deuterium experiments