ABSTRACT
The interaction between DNA and the nonhistone proteins HMGB1 and HMGB1-(A+B) has been studied using circular dichroism and scanning force microscopy. The recombinant protein HMGB1-(A+B) has no negatively charged C-terminal domain characteristic for HMGB1. Our earlier suggestion about the structural interaction of tandem HMGB1-domains of the recombinant protein with DNA was confirmed. It was shown that the C-terminal part modulates the interactions of HMGB1-domains with DNA. Without the C-terminal sequence, the HMGB1-(A+B) protein forms DNA-protein complexes with the ordered supramolecular structure.
Subject(s)
DNA/chemistry , HMGB1 Protein/chemistry , Animals , Cattle , Circular Dichroism , HMGB1 Protein/ultrastructure , Humans , Microscopy, Electron, Scanning , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/ultrastructureABSTRACT
In the development of biosensors, it is essential to understand how the signal-transducing element may perturb surface-bound proteins and nucleic acids. The tip of the atomic force microscope is such an element in atomic force microscopy. In this paper, we describe the influence of tip-sample interactions on the measured height of the DNA repair protein, Ku, that has been adsorbed onto a mica surface which was submerged in aqueous solution. We find that the measured height of the Ku molecule depends critically on whether or not it is associated with DNA. Additionally, we observed that the conditions (time and concentration) under which Ku is incubated with DNA, affect the appearance (number and type) of the DNA-Ku complexes observed.
Subject(s)
Biosensing Techniques/methods , DNA Helicases/chemistry , DNA Helicases/ultrastructure , Microscopy, Atomic Force/methods , Plasmids/chemistry , Plasmids/ultrastructure , Water/chemistry , Biosensing Techniques/instrumentation , Coated Materials, Biocompatible/analysis , Coated Materials, Biocompatible/chemistry , DNA Helicases/analysis , DNA-Binding Proteins/analysis , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/ultrastructure , Image Interpretation, Computer-Assisted/methods , Ku Autoantigen , Nucleic Acid Conformation , Plasmids/analysis , Protein Binding , Protein Conformation , Surface Properties , Water/analysisABSTRACT
Under physiological conditions, multicomponent biological membranes undergo structural changes which help define how the membrane functions. An understanding of biomembrane structure-function relations can be based on knowledge of the physical and chemical properties of pure phospholipid bilayers. Here, we have investigated phase transitions in dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC) bilayers. We demonstrated the existence of several phase transitions in DPPC and DOPC mica-supported bilayers by both atomic force microscopy imaging and force measurements. Supported DPPC bilayers show a broad L(beta)-L(alpha) transition. In addition to the main transition we observed structural changes both above and below main transition temperature, which include increase in bilayer coverage and changes in bilayer height. Force measurements provide valuable information on bilayer thickness and phase transitions and are in good agreement with atomic force microscopy imaging data. A De Gennes model was used to characterize the repulsive steric forces as the origin of supported bilayer elastic properties. Both electrostatic and steric forces contribute to the repulsive part of the force plot.
Subject(s)
1,2-Dipalmitoylphosphatidylcholine/chemistry , Membranes/chemistry , Phase Transition , Phospholipids/chemistry , Transition Temperature , Aluminum Silicates/chemistry , Microscopy, Atomic ForceABSTRACT
We have used magnetic alternating current mode atomic force microscopy (MAC-AFM) to investigate the formation of supported phospholipid bilayers (SPB) by the method of vesicle fusion. The systems studied were dioleoylphosphatidylcholine (DOPC) on mica and mica modified with 3-aminopropyl-triethoxy-silane (APTES), and DOPC vesicles with gramicidin incorporated on mica and APTES-modified mica. The AFM images reveal three stages of bilayer formation: localized disklike features that are single bilayer footprints of the vesicles, partial continuous coverage, and finally complete bilayer formation. The mechanism of supported phospholipid bilayers formation is the fusion of proximal vesicles, rather than surface disk migration. This mechanism does not appear to be affected by incorporation of gramicidin or by surface modification. Once formed, the bilayer develops circular defects one bilayer deep. These defects grow in size and number until a dynamic equilibrium is reached.
