Lía I. Pietrasanta

Probing protein-protein interactions in real time.

We have used a prototype small cantilever atomic force microscope to observe, in real time, the interactions between individual protein molecules. In particular, we have observed individual molecules of the chaperonin protein GroES binding to and then dissociating from individual GroEL proteins, which were immobilized on a mica support. This work suggests that the small cantilever atomic force microscope is a useful tool for studying protein dynamics at the single molecule level.

Fast imaging and fast force spectroscopy of single biopolymers with a new atomic force microscope designed for small cantilevers

Small cantilevers allow for faster imaging and faster force spectroscopy of single biopolymers than previously possible because they have higher resonant frequencies and lower coefficients of viscous damping. We have used a new prototype atomic force microscope with small cantilevers to produce stable tapping-mode images (1 μm×1 μm) in liquid of DNA adsorbed onto mica in as little as 1.7 s per image. We have also used these cantilevers to observe the forced unfolding of individual titin molecules on a time scale an order of magnitude faster than previously reported. These experiments demonstrate that a new generation of atomic force microscopes using small cantilevers will enable us to study biological processes with greater time resolution. Furthermore, these instruments allow us to narrow the gap in time between results from force spectroscopy experiments and molecular dynamics calculations.

Atomic force microscopy and other scanning probe microscopies.

The highlight of the past year is the unfolding and refolding of the muscle protein titin in the atomic force microscope. A related highlight in the intersection between experiment and theory is a recent review of the effects of molecular forces on biochemical kinetics. Other advances in scanning probe microscopy include entropic brushes, molecular sandwiches and applications of atomic force microscopy to gene therapy.

Probing biopolymers with the atomic force microscope: A review

This short review presents an overview of atomic force microscopy (AFM) of biopolymers and specific examples of some of the biopolymers that have been analyzed by AFM. These specific examples include extracellular polymeric substances on the surfaces of bacterial biofilms, condensed DNA, DNA constructs, and DNA-protein interactions. In addition, two examples are presented for AFM analyses of proteins: laminin flexing its arms in solution and neurofilaments entropically brushing away the space around themselves.

Light regulates attachment, exopolysaccharide production, and nodulation in Rhizobium leguminosarum through a LOV-histidine kinase photoreceptor

Rhizobium leguminosarum is a soil bacterium that infects root hairs and induces the formation of nitrogen-fixing nodules on leguminous plants. Light, oxygen, and voltage (LOV)-domain proteins are blue-light receptors found in higher plants and many algae, fungi, and bacteria. The genome of R. leguminosarum bv. viciae 3841, a pea-nodulating endosymbiont, encodes a sensor histidine kinase containing a LOV domain at the N-terminal end (R-LOV-HK). R-LOV-HK has a typical LOV domain absorption spectrum with broad bands in the blue and UV-A regions and shows a truncated photocycle. Here we show that the R-LOV-HK protein regulates attachment to an abiotic surface and production of flagellar proteins and exopolysaccharide in response to light. Also, illumination of bacterial cultures before inoculation of pea roots increases the number of nodules per plant and the number of intranodular bacteroids. The effects of light on nodulation are dependent on a functional lov gene. The results presented in this work suggest that light, sensed by R-LOV-HK, is an important environmental factor that controls adaptive responses and the symbiotic efficiency of R. leguminosarum.

Recent Highlights from Atomic Force Microscopy of DNA

Abstract Seven recent highlights are presented from atomic force microscopy (AFM) of DNA in this lab. The first two involve advances in the observation of enzymatic reactions in near-physiological solutions. E. coli RNA polymerase was observed to process along its DNA template in a series of time-lapse images [S. Kasas, et al., Biochemistry 36, 461 (1997)], and a new small-cantilever atomic force microscope (AFM) imaged DNA degradation by DNase I at rates as fast as two seconds per image. The next five highlights involve structural observations of DNA and DNA-protein complexes, including DNA condensed for gene delivery, sequence-dependent DNA condensation, an AFM assay for RNA polymerase, and AFM evidence for a yeast kinetochore complex that may be involved in holding together sister chromatids during cell division.