Specific DNA-protein interactions on mica investigated by atomic force microscopy.

DNA processing by site-specific proteins on surface remains a challenging issue for nanobioscience applications and, in particular, for high-resolution imaging by atomic force microscopy (AFM). To obtain high-resolution conditions, mica, an atomically flat and negatively charged surface, is generally used. However, even though many specific DNA/protein interactions have already been observed by AFM, little is known about DNA accessibility to specific enzymes on mica.

Atomic force microscopy imaging of DNA under macromolecular crowding conditions.

Studying the influence of macromolecular crowding at high ionic strengths on assemblies of biomolecules is of particular interest because these are standard intracellular conditions. However, up to now, no techniques offer the possibility of studying the effect of molecular crowding at the single molecule scale and at high resolution. We present a method to observe double-strand DNA under macromolecular crowding conditions on a flat mica surface by atomic force microscope.

The EcoRI-DNA complex as a model for investigating protein-DNA interactions by atomic force microscopy.

Atomic force microscopy (AFM) is a technique widely used to image protein-DNA complexes, and its application has now been extended to the measurements of protein-DNA binding constants and specificities. However, the spreading of the protein-DNA complexes on a flat substrate, generally mica, is required prior to AFM imaging. The influence of the surface on protein-DNA interactions is therefore an issue which needs to be addressed.

Anionic polyelectrolyte adsorption on mica mediated by multivalent cations: a solution to DNA imaging by atomic force microscopy under high ionic strengths.

Adsorption of DNA molecules on mica, a highly negatively charged surface, mediated by divalent or trivalent cations is considered. By analyzing atomic force microscope (AFM) images of DNA molecules adsorbed on mica, phase diagrams of DNA molecules interacting with a mica surface are established in terms of concentrations of monovalent salt (NaCl) and divalent (MgCl2) or multivalent (spermidine, cobalt hexamine) salts. These diagrams show two transitions between nonadsorption and adsorption.

Structures of in vitro evolved binding sites on neocarzinostatin scaffold reveal unanticipated evolutionary pathways.

We have recently applied in vitro evolution methods to create in Neocarzinostatin a new binding site for a target molecule unrelated to its natural ligand. The main objective of this work was to solve the structure of some of the selected binders in complex with the target molecule: testosterone. Three proteins (1a.

A new approach to DNA bending by polyamines and its implication in DNA condensation.

Polyamines are known to induce dynamical bending of DNA molecules. This mechanism is very important since many DNA binding proteins (DNAse, transcription factor, etc.) exert their action by their ability to bend DNA.

Crystal structure of YHI9, the yeast member of the phenazine biosynthesis PhzF enzyme superfamily.

In the Pseudomonas bacterial genomes, the PhzF proteins are involved in the production of phenazine derivative antibiotic and antifungal compounds. The PhzF superfamily however also encompasses proteins in all genomes from bacteria to eukaryotes, for which no function has been assigned. We have determined the three dimensional crystal structure at 2.

High-throughput crystal-optimization strategies in the South Paris Yeast Structural Genomics Project: one size fits all?

Crystallization has long been regarded as one of the major bottlenecks in high-throughput structural determination by X-ray crystallography. Structural genomics projects have addressed this issue by using robots to set up automated crystal screens using nanodrop technology. This has moved the bottleneck from obtaining the first crystal hit to obtaining diffraction-quality crystals, as crystal optimization is a notoriously slow process that is difficult to automatize.

Crystal structure of the YML079w protein from Saccharomyces cerevisiae reveals a new sequence family of the jelly-roll fold.

We determined the three-dimensional crystal structure of the protein YML079wp, encoded by a hypothetical open reading frame from Saccharomyces cerevisiae to a resolution of 1.75 A. The protein has no close homologs and its molecular and cellular functions are unknown.

Statistical experimental design of protein crystallization screening revisited.

A statistical experimental design approach was used to prepare a set of solutions for the screening of protein crystallization conditions. This approach is shown to be amenable to quantitative evaluation and therefore to the rational optimization of the screening results. All solutions contain a cryoprotectant, thus eliminating the need for subsequent optimization of crystal freezing conditions.

Crystal structure of yeast allantoicase reveals a repeated jelly roll motif.

Allantoicase (EC 3.5.3.

Crystal structure of the YGR205w protein from Saccharomyces cerevisiae: close structural resemblance to E. coli pantothenate kinase.

The protein product of the YGR205w gene of Saccharomyces cerevisiae was targeted as part of our yeast structural genomics project. YGR205w codes for a small (290 amino acids) protein with unknown structure and function. The only recognizable sequence feature is the presence of a Walker A motif (P loop) indicating a possible nucleotide binding/converting function.

Structure of protein phosphatase methyltransferase 1 (PPM1), a leucine carboxyl methyltransferase involved in the regulation of protein phosphatase 2A activity.

The important role of the serine/threonine protein phosphatase 2A (PP2A) in various cellular processes requires a precise and dynamic regulation of PP2A activity, localization, and substrate specificity. The regulation of the function of PP2A involves the reversible methylation of the COOH group of the C-terminal leucine of the catalytic subunit, which, in turn, controls the enzyme's heteromultimeric composition and confers different protein recognition and substrate specificity. We have determined the structure of PPM1, the yeast methyltransferase responsible for methylation of PP2A.

Crystal structure of the bifunctional chorismate synthase from Saccharomyces cerevisiae.

Chorismate synthase (EC 4.2.3.

Crystal structure of the yeast Phox homology (PX) domain protein Grd19p complexed to phosphatidylinositol-3-phosphate.

Phox homology (PX) domains have been recently identified in a number of different proteins and are involved in various cellular functions such as vacuolar targeting and membrane protein trafficking. It was shown that these modules of about 130 amino acids specifically binding to phosphoinositides and that this interaction is crucial for their cellular function. The yeast genome contains 17 PX domain proteins.

A structural genomics initiative on yeast proteins.

A canonical structural genomics programme is being conducted at the Paris-Sud campus area on baker's yeast proteins. Experimental strategies, first results and identified bottlenecks are presented. The actual or potential contributions to the structural genomics of several experimental structure-determination methods are discussed.