Protein rotor

Part 2 The Fi-motor Transmembrane Rotary Motor Drives Rotation of Oil-like Protein Rotor Within an Orange-Shaped, Six-Subunit Protein Structure, Causing ADP and Pi to Form ATP. 

The most oil-like side of the asymmetric protein rotor, rotated by the Fo-motor, begins to oppose one of three symmetrically arranged catalytic sections that contains ADP and P,. 

Fig. 8.3 Calculated conformational energy display values less than 2kcalmol ligands penalties of the protein-bound ligands 1-36. with five to eight rotors display values less The energy penalty increases with the number than 4kcalmol and ligands with eight to 11 of rotors. Ligands with one to four rotors rotors display values less than 6kcal mol. ...

Applications of Molecular Rotors in Protein Sensing and Sensing of Other... 

Most of the molecules introduced in this chapter are hydrophobic. Even those molecules that have been functionalized to improve water-solubility (for example, CCVJ and CCVJ triethyleneglycol ester 43, Fig. 14) contain large hydrophobic structures. In aqueous solutions that contain proteins or other macromolecules with hydrophobic regions, molecular rotors are attracted to these pockets and bind to the proteins. Noncovalent attraction to hydrophobic pockets is associated with restricted intramolecular rotation and consequently increased quantum yield. In this respect, molecular rotors are superior protein probes, because they do not only indicate the presence of proteins (similar to antibody-conjugated fluorescent markers), but they also report a constricted environment and can therefore be used to probe protein structure and assembly. 

Two of the cytoskeletal components, the actin filaments and the microtubules have been studied with molecular rotors. The main component of the actin filaments is the actin protein, a 44 kD molecule found in two forms within the cell the monomeric globulin form (G-actin) and the filament form (F-actin). Actin binds with ATP to form the microfilaments that are responsible for cell shape and motility. The rate of polymerization from the monomeric form plays a vital role in cell movement and signaling. Actin filaments form the cortical mesh that is the basis of the cytoskeleton. The cytoskeleton has an active relationship with the plasma membrane. Functional proteins found in both structures... 

Akers WJ, Cupps JM, Haidekker MA (2005) Interaction of fluorescent molecular rotors with blood plasma proteins. Biorheology 42(5) 335-344... 

Transfer powder into a 50-ml Falcon-style tube, measure the volume of powder, and add an equal volume of lysis buffer to suspend cell proteins in buffer. Centrifuge at 3500 Xg (4000 rpm in a Sigma 4K15 centrifuge, rotor 11150) for 5 min at 4° to pellet the cell debris. 

The WCE is clarified by two successive centrifugations at 16,100 for 2 and 10 min at 4°, respectively, using an Eppendorf F 45-24-11 rotor in a table centrifuge. After each centrifugation, the supernatant is carefully transferred to a new precooled Eppendorf tube, avoiding the lipid layer, and the total protein concentration (mg/ml) is estimated using the Bradford method (Biorad). 

Approximately 1 mg of total protein is pre-incubated with 25 /il of A-Sepharose beads CL-4B (Amersham Pharmacia Biotech) in 300 /d of binding buffer FLA for 1 h at 4° with gende rocking. The sample is then spun down at 1000 rpm for 3 min in an Eppendorf F 45-24-11 rotor, preserving the supernatant for further use. An aliquot of 3% of the total protein from each reaction is stored at —20° to provide the input reference sample in the subsequent analysis. 

The bound proteins attached to the beads are then spun down at 1000 rpm for 3 min in an Eppendorf F 45-24-11 rotor (preserving a 3% aliquot), and the beads are washed 3 times with 1 ml of the F buffer each. 

DPDPB has been used to study the endocytosis of cadherin from intracellular junctions (Troyanovsky et al., 2006), the subunit arrangement in the flagellar rotor assembly (Lowder et al., 2005), and the disease-associated mutations in myelin proteolipid protein in the endoplasmic reticulum (ER) (Swanton et al., 2005). DPDPB can be used to conjugate reduced antibody molecules to p-D-galactosidase using essentially the same protocol as that described by O Sullivan et al. (1979). 

Figure 5.65 provides theoretical evidence that resonance-assisted H-bonding can serve as an effective mechanism for switching a methyl rotor from one preferred conformation to another, or for controlling the stiffness of torsional motions in alkylated amides. In particular, the torsional potentials of proteins (more specifically, the Ramachandran b angle at Ca) should be sensitive to N—H- O and related H-bonding interactions involving the amide backbone. In principle, this electronic... 

Zonal techniques may be used for the separation of a wide range of particles and macromolecules, e.g. mitochondria, nuclei, ribosomes and proteins. The technique may be used for bulk preparative work using a zonal rotor which is filled with a solvent gradient while running at a slow speed. The sample is similarly introduced and the rotor speed is then increased to the desired value. After centrifugation is complete, the contents are drawn off while the rotor is running slowly by displacing them with a more dense solution. 

A summary of the report follows The problem is to separate proteins. Furthermore, SpinPro should pay particular attention to the purity of the separation. The sample is not negatively affected by sucrose, has a sedimentation coefficient of 16 Svedbergs, and is in liquid form of 3 mL and a concentration of 1% w/w. The protein of interest should be placed 45% from the top of the gradient at the end of the run. Of the gradient concentrations 10-40% and 5-20%, the 10-40% is preferred by the investigator. There are no solvents in the sample that are harmful to the tubes. Finally, from the lab, SpinPro should use the L2-75B ultracentrifuge and the SW 41 Ti rotor, which does not require a speed derating due to its age. 

Load 0.3 mL of the Protein sample in full tubes at the top position of the gradient. Applying the correct amount of sample is important to prevent "overloading" the gradient. The rotor tubes can be run full or half full, or bottles can be used in place of tubes. 

Solubilization of Membrane Proteins. A modification of the procedure of Hjelmeland et al. (30) was employed. A 300-g portion of liquid-nitrogen frozen, 6-day Nicotiana silvestris cultured cells was suspended in 200 ml of 50 mM N-(2-hydroxyethyl)-piperazine-N -3-propanesulfonic acid (EPPS-KOH) buffer, 1 mM dithiothreitol (DTT), and 0.1 mM EDTA extraction buffer with constant stirring until completely suspended (20 min). The slurry was centrifuged in a Sorvall SS 34 rotor at 9,000g for 20 min at 4°C. The supernatant was passed through miracloth (Calbiochem). An aliquot... 

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