Organization and dynamics of tryptophan residues in erythroid spectrin: Novel structural features of denatured spectrin revealed by the wavelength‐selective fluorescence approach

Abstract

We have investigated the organization and dynamics of the functionally important tryptophan residues of erythroid spectrin in native and denatured conditions utilizing the wavelength-selective fluorescence approach. We observed a red edge excitation shift (REES) of 4 nm for the tryptophans in the case of spectrin in its native state. This indicates that tryptophans in spectrin are localized in a microenvironment of restricted mobility, and that the regions surrounding the spectrin tryptophans offer considerable restriction to the reorientational motion of the water dipoles around the excited state tryptophans. Interestingly, spectrin exhibits a REES of 3 nm even when denatured in 8 M urea. This represents the first report of a denatured protein displaying REES. Observation of REES in the denatured state implies that some of the structural and dynamic features of this microenvironment around the spectrin tryptophans are retained even when the protein is denatured. Fluorescence quenching data of denatured spectrin support this conclusion. In addition, we have deduced the organization and dynamics of the hydrophobic binding site of the polarity-sensitive fluorescent probe PRODAN that binds erythroid spectrin with high affinity. When bound to spectrin, PRODAN exhibits a REES of 9 nm. Because PRODAN binds to a hydrophobic site in spectrin, such a result would directly imply that this region of spectrin offers considerable restriction to the reorientational motion of the solvent dipoles around the excited state fluorophore. The results of our study could provide vital insight into the role of tryptophans in the stability and folding of spectrin.

Novel Insights Into Protein Structure and Dynamics Utilizing the Red Edge Excitation Shift Approach

“…80 The REES approach has been used in a number of cases to monitor the tryptophan environment and dynamics in proteins such as human α 1 -acid glycoprotein, 81 bothropstoxin-I from the venom of Bothrops jararacussu, 82 ascorbate oxidase, 83 smooth muscle myosin light chain kinase, 84 skeletal myosin rod, 85 the human leukocyte antigen complex, 86 and the pore-forming α-toxin from Staphylococcus aureus. 87 In addition, it has been shown that the environment of tryptophans in cytoskeletal proteins such as tubulin 77 and spectrin 88,89 are motionally restricted and display REES. Importantly, observation of REES in multitryptophan proteins considerably rules out the possibility of homotransfer among tryptophans.…”

“…77 In a recent study on the cytoskeletal protein erythroid spectrin, the tryptophans show REES indicating the localization in environments which are motionally restricted due to slow solvent relaxation. 88,89 Interestingly, spectrin display REES even when denatured in 8 M urea. 88 This surprising result is in contrast to earlier studies where it was shown that the emission maximum of tryptophans in denatured proteins do not exhibit excitation wavelength dependence.…”

“…88,89 Interestingly, spectrin display REES even when denatured in 8 M urea. 88 This surprising result is in contrast to earlier studies where it was shown that the emission maximum of tryptophans in denatured proteins do not exhibit excitation wavelength dependence. This is because the tryptophans are exposed to water when denatured and therefore do not offer any restriction to the solvent (water) dipoles around them in the excited state.…”

“…This is further supported by analysis of fluorescence quenching data using acrylamide as quencher. 88 Such residual structure in an unfolded protein is thought to reside predominantly in hydrophobic clusters, where residues like tryptophan stabilize these networks through cooperative long range nonnative interactions. 95 Although residual structure in denatured proteins has been studied before, 96,97 this example constitutes the first demonstration of slow solvent relaxation in a completely denatured protein.…”

“…182 The organization and dynamics of the PRODAN binding site in erythroid spectrin have been monitored recently. 88 The observation of REES for spectrin-bound PRODAN suggests that PRODAN is in an environment where its mobility is considerably reduced. Since PRODAN binds to the hydrophobic site in spectrin, this result shows that this region offers considerable restriction to the reorientational motion of the solvent dipoles around the excited state fluorophore.…”