Correlation of membrane protein conformational and functional dynamics.

Conformational changes in ion channels lead to gating of an ion-conductive pore. Ion flux has been measured with high temporal resolution by single-channel electrophysiology for decades. However, correlation between functional and conformational dynamics remained difficult, lacking experimental techniques to monitor sub-millisecond conformational changes.

Direct visualization of the Sec translocase engaging precursor proteins in lipid bilayers.

exports proteins via a translocase comprising SecA and the translocon, SecYEG. Structural changes of active translocases underlie general secretory system function, yet directly visualizing dynamics has been challenging. We imaged active translocases in lipid bilayers as a function of precursor protein species, nucleotide species, and stage of translocation using atomic force microscopy (AFM).

Quiet Outer Membrane Protein G (OmpG) Nanopore for Biosensing.

Interest in nanopore technology has been growing due to nanopores' unique capabilities in small molecule sensing, measurement of protein folding, and low-cost DNA and RNA sequencing. The E. coli β-barrel outer membrane protein OmpG is an excellent alternative to other protein nanopores because of its single polypeptide chain.

Quaternary structure of the small amino acid transporter OprG from .

is an opportunistic human pathogen that causes nosocomial infections. The outer membrane contains specific porins that enable substrate uptake, with the outer membrane protein OprG facilitating transport of small, uncharged amino acids. However, the pore size of an eight-stranded β-barrel monomer of OprG is too narrow to accommodate even the smallest transported amino acid, glycine, raising the question of how OprG facilitates amino acid uptake.

The Hessian Blob Algorithm: Precise Particle Detection in Atomic Force Microscopy Imagery.

Imaging by atomic force microscopy (AFM) offers high-resolution descriptions of many biological systems; however, regardless of resolution, conclusions drawn from AFM images are only as robust as the analysis leading to those conclusions. Vital to the analysis of biomolecules in AFM imagery is the initial detection of individual particles from large-scale images. Threshold and watershed algorithms are conventional for automatic particle detection but demand manual image preprocessing and produce particle boundaries which deform as a function of user-defined parameters, producing imprecise results subject to bias.

Glass is a Viable Substrate for Precision Force Microscopy of Membrane Proteins.

Though ubiquitous in optical microscopy, glass has long been overlooked as a specimen supporting surface for high resolution atomic force microscopy (AFM) investigations due to its roughness. Using bacteriorhodopsin from Halobacterium salinarum and the translocon SecYEG from Escherichia coli, we demonstrate that faithful images of 2D crystalline and non-crystalline membrane proteins in lipid bilayers can be obtained on microscope cover glass following a straight-forward cleaning procedure. Direct comparison between AFM data obtained on glass and on mica substrates show no major differences in image fidelity.

Transient collagen triple helix binding to a key metalloproteinase in invasion and development.

Skeletal development and invasion by tumor cells depends on proteolysis of collagen by the pericellular metalloproteinase MT1-MMP. Its hemopexin-like (HPX) domain binds to collagen substrates to facilitate their digestion. Spin labeling and paramagnetic nuclear magnetic resonance (NMR) detection have revealed how the HPX domain docks to collagen I-derived triple helix.

Heparinoids activate a protease, secreted by mucosa and tumors, via tethering supplemented by allostery.

Activation by glycosaminoglycans (GAGs) is an emerging trend among extracellular proteases important in disease. ProMMP-7, the zymogen of a matrix metalloproteinase secreted by mucosal epithelial and tumor cells, is activated at their surfaces by sulfated GAGs, but how? ProMMP-7 is activated in trans by representative heparin oligosaccharides in a length-dependent manner, with a large jump in activation at lengths of 16 monosaccharides. Imaging by atomic force microscopy visualized small complexes of proMMP-7 molecules linked by 8-mer lengths of heparinoids and extended assembles formed with 16-mer lengths of heparin.

Stoichiometry of SecYEG in the active translocase of Escherichia coli varies with precursor species.

We have established a reconstitution system for the translocon SecYEG in proteoliposomes in which 55% of the accessible translocons are active. This level corresponds to the fraction of translocons that are active in vitro when assessed in their native environment of cytoplasmic membrane vesicles. Assays using these robust reconstituted proteoliposomes and cytoplasmic membrane vesicles have revealed that the number of SecYEG units involved in an active translocase depends on the precursor undergoing transfer.

Dynamic structure of the translocon SecYEG in membrane: direct single molecule observations.

Purified SecYEG was reconstituted into liposomes and studied in near-native conditions using atomic force microscopy. These SecYEG proteoliposomes were active in translocation assays. Changes in the structure of SecYEG as a function of time were directly visualized.