Measurement of intrinsic fluorescence to probe the conformational flexibility and thermodynamic stability of a single tryptophan protein entrapped in a sol–gel derived glass matrix

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Abstract
In this study techniques based on measurement of intrinsic tryptophan (Trp) fluorescence were used to monitor the stability and conformational flexibility of the single tryptophan protein monellin which was entrapped in sol–gel derived glass monoliths formed from tetraethylorthosilicate. The monoliths were aged either in buffer (wet-aged) or in air (dry-aged). The steady-state fluorescence spectra, steady-state anisotropy and acrylamide quenching behaviour of the single Trp at position 3 were monitored during chemical denaturation with guanidine hydrochloride, and during thermal denaturation. These studies indicated that there were no significant improvements in either chemical or thermal stability when the protein was present in wet-aged monoliths. However, the long-term stability of the protein was improved six-fold when such monoliths were stored at 4 °C. The encapsulation of monellin into dry-aged monoliths caused the thermal unfolding transition to broaden and shift upward by 10 °C, and caused the long-term stability to improve by 12-fold (compared to solution). Chemical stability studies also showed a broader transition for the unfolding of the protein in dry-aged monoliths, and suggested that the protein was present in a distribution of environments, each with a slightly different thermodynamic stability. The steady-state fluorescence responses obtained during denaturation and the accessibility of native and denatured protein to quencher provided clear evidence that the entrapped protein had a smaller range of conformational motions compared to the protein in solution, and that the entrapped protein was not able to unfold completely. The restriction of conformational motion, along with the increased structural order of the internal environment of the monoliths, play a role in the improvements in thermal and long-term stability that were observed.