We have investigated the folding dynamics of Thermus thermophilus cytochrome c 552 by time-resolved fluorescence energy transfer between the heme and each of seven site-specific fluorescent probes. We have found both an equilibrium unfolding intermediate and a distinct refolding intermediate from kinetics studies. Depending on the protein region monitored, we observed either two-state or three-state denaturation transitions. The unfolding intermediate associated with three-state folding exhibited native contacts in β-sheet and C-terminal helix regions. We probed the formation of a refolding intermediate by time-resolved fluorescence energy transfer between residue 110 and the heme using a continuous flow mixer. The intermediate ensemble, a heterogeneous mixture of compact and extended polypeptides, forms in a millisecond, substantially slower than the ∼100-μs formation of a burst-phase intermediate in cytochrome c. The surprising finding is that, unlike for cytochrome c, there is an observable folding intermediate, but no microsecond burst phase in the folding kinetics of the structurally related thermostable protein.FRET | microfluidic mixing | protein refolding

Background:To evaluate the change in intraocular pressure (IOP) in patients with idiopathic intracranial hypertension (IIH) who underwent optic nerve sheath fenestration (ONSF) and to determine if radiographic evidence of posterior scleral or globe indentation influenced IOP.Methods:This is a retrospective analysis of IOP in IIH patients who underwent ONSF. The study included all patients from September 2010 to March 2018 operated on by a single surgeon (R.M.). IOPs preoperatively and postoperatively were recorded along with the acetazolamide dosage and whether there was evidence of posterior scleral or globe indentation on preoperative MRI.Results:A total of 29 patients (35 eyes) with IIH underwent ONSF. The average reduction in IOP among all patients was 1.24 mm Hg (P = 0.0218), but this increased to 2.69 mm Hg (P = 0.004) in patients who were maintained on the same dosage of acetazolamide in the preoperative and postoperative period. Furthermore, the reduction in IOP in those with posterior scleral or globe indentation was 2.5 mm Hg (P = 0.0095). When the perioperative period was evaluated, the mean decrease in IOP was 1.83 mm Hg (P = 0.0217).Conclusions:Reducing the cerebral spinal fluid pressure (CSFP) at the level of the intraorbital optic nerve through an ONSF can slightly reduce the IOP. In those with evidence of posterior globe or scleral indentation/flattening, the reduction in IOP was higher, which supports the theory that CSF pressure indents the globe and leads to an increase in IOP. Although these changes in IOP are small, this study provides further evidence for a connection between IOP and CSFP.

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The inhomogeneous alloy broadening of deep levels produced by substitutional impurities in crystalhne semiconductor alloys is investigated with use of a technique based upon the embedded-cluster method and the theory of deep levels. Results are presented for the composition dependences of the center, width, and component lines of the alloy-broadened spectrum of nitrogen on the arsenic site in Al Ga~"As. For the present application, only the effects of the alloy disorder in the nearestneighbor environment of the impurity are accounted for by the theory. In principle, however, the method outlined here is generalizable to account for such effects in the impurity s secondand more-distant-neighbor environments,