Risiko- und Finanzierungspolitik im Rahmen einer Ausweitung der Aussenhandelstaetigkeit auf den osteuropaeischen Markt

SIGLEAvailable from Bibliothek des Instituts fuer Weltwirtschaft, ZBW, Duesternbrook Weg 120, D-24105 Kiel W 201 (258) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekDEGerman

Cooperative Binding of PhoB^DBD to Its Cognate DNA SequenceA Combined Application of Single-Molecule and Ensemble Methods

A combined approach based on isothermal titration calorimetry (ITC), fluorescence resonance energy transfer (FRET) experiments, circular dichroism spectroscopy (CD), atomic force microscopy (AFM) dynamic force spectroscopy (DFS), and surface plasmon resonance (SPR) was applied to elucidate the mechanism of protein–DNA complex formation and the impact of protein dimerization of the DNA-binding domain of PhoB (PhoB^DBD). These insights can be translated to related members of the family of winged helix-turn-helix proteins. One central question was the assembly of the trimeric complex formed by two molecules of PhoB^DBD and two cognate binding sites of a single oligonucleotide. In addition to the native protein WT-PhoB^DBD, semisynthetic covalently linked dimers with different linker lengths were studied. The ITC, SPR, FRET, and CD results indicate a positive cooperative binding mechanism and a decisive contribution of dimerization on the complex stability. Furthermore, an alanine scan was performed and binding of the corresponding point mutants was analyzed by both techniques to discriminate between different binding types involved in the protein–DNA interaction and to compare the information content of the two methods DFS and SPR. In light of the published crystal structure, four types of contribution to the recognition process of the <i>pho</i> box by the protein PhoB^DBD could be differentiated and quantified. Consequently, it could be shown that investigating the interactions between DNA and proteins with complementary techniques is necessary to fully understand the corresponding recognition process

Cooperative Binding of PhoB(DBD) to Its Cognate DNA Sequence-A Combined Application of Single-Molecule and Ensemble Methods

Ritzefeld M, Walhorn V, Kleineberg C, et al. Cooperative Binding of PhoB(DBD) to Its Cognate DNA Sequence-A Combined Application of Single-Molecule and Ensemble Methods. Biochemistry. 2013;52(46):8177-8186.A combined approach based on isothermal titration calorimetry (ITC), fluorescence resonance energy transfer (FRET) experiments, circular dichroism spectroscopy (CD), atomic force microscopy (AFM) dynamic force spectroscopy (DFS), and surface plasmon resonance (SPR) was applied to elucidate the mechanism of protein-DNA complex formation and the impact of protein dimerization of the DNA-binding domain of PhoB (PhoB(DBD)). These insights can be translated to related members of the family of winged helix-turn-helix proteins. One central question was the assembly of the trimeric complex formed by two molecules of PhoB(DBD) and two cognate binding sites of a single oligonucleotide. In addition to the native protein WT-PhoB(DBD), semisynthetic covalently linked dimers with different linker lengths were studied. The ITC, SPR, FRET, and CD results indicate a positive cooperative binding mechanism and a decisive contribution of dimerization on the complex stability. Furthermore, an alanine scan was performed and binding of the corresponding point mutants was analyzed by both techniques to discriminate between different binding types involved in the protein-DNA interaction and to compare the information content of the two methods DFS and SPR. In light of the published crystal structure, four types of contribution to the recognition process of the pho box by the protein PhoB(DBD) could be differentiated and quantified. Consequently, it could be shown that investigating the interactions between DNA and proteins with complementary techniques is necessary to fully understand the corresponding recognition process

The Kruppel-like factor KLF4 is a critical regulator of monocyte differentiation

Monocyte differentiation involves the participation of lineage-restricted transcription factors, although the mechanisms by which this process occurs are incompletely defined. Within the hematopoietic system, members of the Kruppel-like family of factors (KLFs) play essential roles in erythrocyte and T lymphocyte development. Here we show that KLF4/GKLF is expressed in a monocyte-restricted and stage-specific pattern during myelopoiesis and functions to promote monocyte differentiation. Overexpression of KLF4 in HL-60 cells confers the characteristics of mature monocytes. Conversely, KLF4 knockdown blocked phorbol ester-induced monocyte differentiation. Forced expression of KLF4 in primary common myeloid progenitors (CMPs) or hematopoietic stem cells (HSCs) induced exclusive monocyte differentiation in clonogenic assays, whereas KLF4 deficiency inhibited monocyte but increased granulocyte differentiation. Mechanistic studies demonstrate that KLF4 is a target gene of PU.1. Consistently, KLF4 can rescue PU.1−/− fetal liver cells along the monocytic lineage and can activate the monocytic-specific CD14 promoter. Thus, KLF4 is a critical regulator in the transcriptional network controlling monocyte differentiation