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We find that the isolated, extracellular domain of tissue factor (TF1-218; sTF) exhibits only 4% of the activity of wild-type transmembrane TF (TF1-263) in an assay that measures the conversion of factor X to Xa by the TF:VIIa complex. Further, the activity of sTF is manifest only when vesicles consisting of phosphatidylserine and phosphatidylcholine (30/70 w/w) are present. To determine whether the decreased activity results from weakened affinity of sTF for VIIa, we studied their interaction using equilibrium ultracentrifugation, fluorescence anisotropy, and an activity titration. Ultracentrifugation of the sTF:VIIa complex established a stoichiometry of 1:1 and an upper limit of 1 nM for the equilibrium dissociation constant (Kd). This value is in agreement with titrations of dansyl-D-Phe-L-Phe-Arg chloromethyl ketone active site labeled VIIa (DF-VIIa) with sTF using dansyl fluorescence anisotropy as the observable. Pressure dissociation experiments were used to obtain quantitative values for the binding interaction. These experiments indicate that the Kd for the interaction of sTF with DF-VIIa is 0.59 nM (25 degrees C). This value may be compared to a Kd of 7.3 pM obtained by the same method for the interaction of DF-VIIa with TF1-263 reconstituted into phosphatidylcholine vesicles. The molar volume change of association was found to be 63 and 117 mL mol-1 for the interaction of DF-VIIa with sTF and TF1-263, respectively. These binding data show that the sTF:VIIa complex is quantitatively and qualitatively different from the complex formed by TF1-263 and VIIa.

Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators OR and OL controls the developmental state of the phage. It has long been believed that cooperativity is mediated by self-assembly of repressor dimers to form tetramers which can then bind simultaneously to adjacent operator sites. As a first step in defining the individual energy contributions to binding cooperativity, sedimentation equilibrium and steady-state fluorescence anisotropy methods have been used to study the higher order assembly reactions of the free repressor in solution. Wild-type repressor with 5-hydroxytryptophan (5-OHTrp) substituted for the native tryptophan [Ross et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12023-12027] and two mutant repressor proteins that bind cooperatively to OR but have altered dimerization properties were also studied. We report here that the primary assembly mode of all four proteins is dimer to octamer. It is not dimer to tetramer as previously assumed. While tetramer does form as an assembly intermediate, dimer-octamer assembly is a concerted process so that tetramer is never a predominant species in solution. Sedimentation velocity experiments suggest that the octamer is highly asymmetric, consistent with an elongated shape. This conformation could allow octamers to bind simultaneously to all three operator sites at either OR or OL. Examination of tetramer and octamer concentrations suggests that both species could be involved in cooperative repressor-operator interactions. Our previous work used the unique spectral properties of 5-OHTrp to demonstrate that octamer binds single-operator DNA and is not dissociated to tetramer [Laue et al. (1993) Biochemistry 32, 2469-2472]. Taken together with the results presented here, octamers as well as tetramers must be considered in developing models to explain the cooperativity of lambda cI repressor binding to operator DNA.

We have used a tryptophan-requiiing Escherichia coli auxotroph to replace the three tryptophan residues of A cI repressor with 5-hydroxy-L-tryptophan (5-OHTrp). By using a nonleaky promoter, we have achieved >95% replacement of tryptophan in the repressor. We show that the absorbance and fluorescence properties of 5-OHTrp-A cI are clearly distinct from A cI repressor and that the fluorescence of 5-OHTrp-A cI repressor can be observed selectively in the presence of exogenous tryptophan. We also show that the 5-OHTrp-A cI repressor functional properties, as assessed by measurement of binding constants for self-association and for association to operator DNA, and structural properties, as assessed by fluorescence, are indistinguishable from the native repressor. Based on these results, we anticipate that the availability of spectrally enhanced proteins wiil significantly enhance the utility of both fluorescence and phosphorescence spectroscopies to study protein structure and function in complex interacting systems.Tryptophan fluorescence has been used widely to study conformation, function, dynamics, and intermolecular interactions of proteins (1). However, the presence of at least one and often several tryptophans in most proteins severely limits the utility oftryptophan fluorescence for studying one protein species in the company of others. For this reason, many fluorescence studies involving proteins and polypeptides make use of extrinsic probes, such as dansyl or fluorescein. Use of extrinsic probes, however, is complicated by the difficulty of specific placement and the risk that chemical modification can alter the functional and structural properties of the labeled protein.Clearly, it would be to great advantage to incorporate into a protein a fluorophore that is spectrally distinct from tryptophan while having none of the disadvantages of extrinsic probes. In this way, a spectrally enhanced protein (SEP) would be generated that would still have "intrinsic" fluorescence. To be useful for the generation of SEPs, a candidate tryptophan analogue mlist be readily incorporated by biosynthesis into the protein ofinterest, must be fluorescent and have spectral properties distinct from those oftryptophan, and must have little or no effect on the functional and structural properties of the protein into which it has been incorporated.An intriguing possibility for creation of SEPs is suggested by NMR studies in which tryptophan auxotrophs of Escherichia coli were used to incorporate fluorotryptophan derivatives into Salmonella typhimurium histidine binding protein J (2) and rat cellular retinol binding protein (3). The fluorotryptophan derivatives, however, are spectrally very similar to tryptophan. Thus, they do not have features that enhance fluorescence studies. By contrast, the absorbance spectra of another group of tryptophan derivatives, the hydroxytryptophans, are sufficiently red-shifted to allow selective excitation in the presence of tryptophan. In addition, several hydroxytryptophan derivatives have ...

The coronavirus disease 2019 pandemic has led to widespread change as public health strategies for containment have emphasized social distancing and remaining at home. These policies have led to downscaled clinic volumes, cancellation of elective procedures, enhanced personal protective strategies in the clinic, and adoption of telemedicine encounters. We describe the evidence-based practical approach taken in our ophthalmology department to continue delivering eye care during the pandemic by rescheduling visits, enhancing clinic safety, and adopting virtual video encounters. Key Summary PointsThis article covers the following topics relating to ophthalmology practice during the coronavirus disease 2019 pandemic.Brief background on COVID-19 and ophthalmic manifestations.Triage and rescheduling clinic appointments and OR procedures.Telemedicine, its role and implementation in the current pandemic. Early teleophthalmology experience from UPMC.This study has two Co-Principal Investigators: