Tracking Structural Changes in the UCS Domain of the Myosin Chaperone UNC-45 by Fluorescence Spectroscopy

Abstract

Molecular chaperones are commonly identified by their ability to suppress heat-induced protein aggregation. The muscle-specific molecular chaperone UNC-45 is known to be involved in myosin folding and is trafficked to the sarcomeres A-band during thermal stress. Using a combination of fluorescence, circular dichroism, limited proteolysis and mass spectroscopy to quantitatively analyze the effect of temperature on UNC-45 we previously found that the UCS domain undergoes significant structural changes in response to temperatures within a heat-shock range (Bujalowski, et al., FEBS let 2015). In order to further analyze the regions within the UCS domain that become exposed to the solvent we used fluorescent assays based on the environment-sensitive hydrophobic fluorescent dye, Bis-ANS. We found that UCS interacts with Bis-ANS in 2:1 stoichiometry at 25oC with an apparent Kd of 46nM. We found that the number of bound ANS molecules increases ∼10-fold when exposed to temperatures above 35oC. We found that Bis-ANS stably binds to the UCS domain after heating (indicated by the asymmetry in the heating and cooling curves). We further investigated temperature-induced structural changes tryptophan fluorescence. The UCS domain has three Trp residues, W783, W823 and W863. We found that the relative Trp fluorescence signal decreases of as the temperature was raised indicating a change from an hydrophobic to an aqueous environment. In order to quantify the energetics of the ANS-UCS interaction we used isothermal titration calorimetry (ITC). Analysis of the calorimetric titration profile showed a monotonic decrease in the exothermic heat of binding. From these data we estimated an N= 4 (number of binding sites) and DS= + 70 cal/mol/K, suggesting that hydrophobic forces are dominant in the binding of ANS to UCS. In order to approximately map the regions at which ANS binds in the UCS domain we used resonance energy transfer (FRET) between Trp residues and Bis-ANS. We found that as Bis-ANS is added, Trp fluorescence is quenched as energy is transferred to the extrinsic fluorophore in a concentration dependent fashion. We are using these techniques to map the key amino acid regions in the UCS domain that mediate its myosin-binding and chaperoning functions. This work was supported by the American Heart Foundation (AHA 13GRNT17290006) and the NIH (1R01GM118534 01).