High-throughput mechanobiology: Force modulation of ensemble biochemical and cell-based assays.

Mechanobiology is focused on how the physical forces and mechanical properties of proteins, cells, and tissues contribute to physiology and disease. Although the response of proteins and cells to mechanical stimuli is critical for function, the tools to probe these activities are typically restricted to single-molecule manipulations. Here, we have developed a novel microplate reader assay to encompass mechanical measurements with ensemble biochemical and cellular assays, using a microplate lid modified with magnets.

Rapid folding of the prion protein captured by pressure-jump.

The conversion of the cellular form of the prion protein (PrP(C)) to an altered disease state, generally denoted as scrapie isoform (PrP(Sc)), appears to be a crucial molecular event in prion diseases. The details of this conformational transition are not fully understood, but it is perceived that they are associated with misfolding of PrP or its incapacity to maintain the native fold during its cell cycle. Here we present a tryptophan mutant of PrP (F198W), which has enhanced fluorescence sensitivity to unfolding/refolding transitions.

Fast pressure jumps can perturb calcium and magnesium binding to troponin C F29W.

We have used rapid pressure jump and stopped-flow fluorometry to investigate calcium and magnesium binding to F29W chicken skeletal troponin C. Increased pressure perturbed calcium binding to the N-terminal sites in the presence and absence of magnesium and provided an estimate for the volume change upon calcium binding (-12 mL/mol). We observed a biphasic response to a pressure change which was characterized by fast and slow reciprocal relaxation times of the order 1000/s and 100/s.

Selective perturbation of the myosin recovery stroke by point mutations at the base of the lever arm affects ATP hydrolysis and phosphate release.

After ATP binding the myosin head undergoes a large structural rearrangement called the recovery stroke. This transition brings catalytic residues into place to enable ATP hydrolysis, and at the same time it causes a swing of the myosin lever arm into a primed state, which is a prerequisite for the power stroke. By introducing point mutations into a subdomain interface at the base of the myosin lever arm at positions Lys(84) and Arg(704), we caused modulatory changes in the equilibrium constant of the recovery stroke, which we could accurately resolve using the fluorescence signal of single tryptophan Dictyostelium myosin II constructs.

Reversible movement of switch 1 loop of myosin determines actin interaction.

The conserved switch 1 loop of P-loop NTPases is implicated as a central element that transmits information between the nucleotide-binding pocket and the binding site of the partner proteins. Recent structural studies have identified two states of switch 1 in G-proteins and myosin, but their role in the transduction mechanism has yet to be clarified. Single tryptophan residues were introduced into the switch 1 region of myosin II motor domain and studied by rapid reaction methods.

Cryptococcus neoformans senses CO2 through the carbonic anhydrase Can2 and the adenylyl cyclase Cac1.

Cryptococcus neoformans, a fungal pathogen of humans, causes fatal meningitis in immunocompromised patients. Its virulence is mainly determined by the elaboration of a polysaccharide capsule surrounding its cell wall. During its life, C.

Protonation, photobleaching, and photoactivation of yellow fluorescent protein (YFP 10C): a unifying mechanism.

Yellow fluorescent protein (YFP 10C) is widely used as a probe in biology, but its complex photochemistry gives rise to unusual behavior that requires fuller definition. Here we characterize the kinetics of protonation and reversible bleaching over time scales of picoseconds to hours. Stopped-flow and pressure-jump techniques showed that protonation of the fluorescent YFP(-) anion state is two-step with a slow transition that accounts for blinking of 527 nm emission at the single molecule level on the seconds time scale.

PlasmoDB: the Plasmodium genome resource. A database integrating experimental and computational data.

PlasmoDB (http://PlasmoDB.org) is the official database of the Plasmodium falciparum genome sequencing consortium. This resource incorporates the recently completed P.

A novel pressure-jump apparatus for the microvolume analysis of protein-ligand and protein-protein interactions: its application to nucleotide binding to skeletal-muscle and smooth-muscle myosin subfragment-1.

Reactions involving proteins frequently involve large changes in volume, which allows the equilibrium position to be perturbed by changes in pressure. Rapid changes in pressure can thus be used to initiate relaxation in pressure; however, this approach is seldom used, because it requires specialized equipment. We have built a microvolume (50 microl) pressure-jump apparatus, powered by a piezoelectric actuator, based on the original design of Clegg and Maxfield [(1976) Rev.