Effect of ionic strength on the organization and dynamics of tryptophan residues in erythroid spectrin: A fluorescence approach

Abstract

The ionic strength of the medium plays an important role in the structure and conformation of erythroid spectrin. The spectrin dimer is a flexible rod at physiological ionic strength. However, lower ionic strength results in elongation and rigidification (stiffening) of spectrin as shown earlier by electron microscopy and hydrodynamic studies. The ionic strength induced structural transition does not involve any specific secondary structural changes. In this article, we have used a combination of fluorescence spectroscopic approaches that include red edge excitation shift (REES), fluorescence quenching, time-resolved fluorescence measurements, and chemical modification of the spectrin tryptophans to assess the environment and dynamics of tryptophan residues of spectrin under different ionic strength conditions. Our results show that while REES, fluorescence anisotropy, lifetime, and chemical modification of spectrin tryptophans remain unaltered in low and high ionic strength conditions, quenching of tryptophan fluorescence by the aqueous quencher acrylamide (but not the hydrophobic quencher trichloroethanol) and resonance energy transfer to a dansyl-labeled fatty acid show differences in tryptophan environment. These results, which report tertiary structural changes in spectrin upon change in ionic strength, are relevant in understanding the molecular details underlying the conformational flexibility of spectrin.

Effect of ionic strength on folding and aggregation of the hemolytic peptide melittin in solution

“…REES represents a powerful approach that can be used to directly monitor the environment and dynamics around a fluorophore in a complex biological system. 25,26 We have previously shown that REES serves as a sensitive tool to monitor the organization and dynamics of peptides and proteins in solution [27][28][29] and when bound to membranes and membrane-mimetic systems. [9][10][11][12][13]30,31 In this paper, we demonstrate for the first time that the information on the dynamics of hydration obtained by REES could be used as a powerful tool to monitor the self-association of melittin in solution.…”

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.…”

Spectrin Organization and Dynamics: New Insights

“…A plot of the ratio of (I 1 /I 3 ) of 0.2 lM pyrene fluorescence as a function of increasing spectrin concentration is shown in (c). The plots in (b) and (c) are adapted and modified from Haque et al (2000) distributed over the entire molecule and yet are localized in the same position in each domain makes them convenient intrinsic fluorescence reporter groups for monitoring conformational changes in spectrin that contribute to its elastic deformability exhibited in physiological conditions (Subbarao and MacDonald 1994;Kelkar et al 2005). Many of these tryptophans are at or in the vicinity of hydrophobic patches in spectrin, which can bind hydrophobic ligands such as fatty acids and phospholipids and cause quenching of tryptophan fluorescence (Sikorski et al 1987;Kahana et al 1992).…”

“…Each repeat unit (~23 in a and 17 in b) can independently fold to form a triple helical coiled coil (see insets a and b). Adapted and modified from Kelkar et al (2005). Inset (b) shows a representation of the solution NMR structure of the spectrin repeat (Pascual et al 1997) with the tryptophan residues highlighted, drawn using RASMOL ver.…”