Effect of denaturants on the structure and activity of 3-hydroxybenzoate-6-hydroxylase

The effect of denaturants such as urea, sodium dodecylsulphate (SDS), guanidinium hydrochloride (Gu.HCl) on the structure of enzyme 3-hydroxybenzoate-6-hydroxylase was studied using intrinsic fluorescence and far and near-UV-CD spectroscopic techniques. Also, activity profiles of the enzyme, as a function of increasing concentrations of denaturants were studied. The far-UV CD spectrum of the enzyme did not show appreciable alterations in the presence of urea, SDS or Gu.HCl, thereby suggesting that the protein does not undergo gross conformational changes in its alpha-helical secondary structure. The treatment of enzyme with 2 M urea resulted in almost complete loss of catalytic activity, accompanied by the reduction of emission fluorescence of enzyme. Similarly, treatment with 0.01% SDS also caused almost complete loss of activity and quenching of enzyme fluorescence as well as a red shift in the emission peak. In addition, reduction in the intensity of near-UV-CD spectrum, especially at 280 nm was observed. About 70% of the activity was lost by treatment with 20 mM Gu.HCl, accompanied by quenching of intrinsic fluorescence of the enzyme. The change in intrinsic fluorescence of the enzyme in the presence of 5 mM-100 mM Gu.HCI could be correlated to progressive loss of catalytic activity. Thus, intrinsic fluorescence (due to tryptophan residues) could be used as an effective probe to provide an insight into the relation between the activity and subtle conformational changes of the enzyme. The results suggested that denaturants caused very slight conformational changes in the enzyme that perturbed the microenvironment of aromatic amino acid residues such as tryptophan accompanied by reduction or loss of catalytic activity.     

Probing Conformational Stability and Dynamics of Erythroid and Nonerythroid Spectrin: Effects of Urea and Guanidine Hydrochloride

We have studied the conformational stability of the two homologous membrane skeletal proteins, the erythroid and non-erythroid spectrins, in their dimeric and tetrameric forms respectively during unfolding in the presence of urea and guanidine hydrochloride (GuHCl). Fluorescence and circular dichroism (CD) spectroscopy have been used to study the changes of intrinsic tryptophan fluorescence, anisotropy, far UV-CD and extrinsic fluorescence of bound 1-anilinonapthalene-8-sulfonic acid (ANS). Chemical unfolding of both proteins were reversible and could be described as a two state transition. The folded erythroid spectrin and non-erythroid spectrin were directly converted to unfolded monomer without formation of any intermediate. Fluorescence quenching, anisotropy, ANS binding and dynamic light scattering data suggest that in presence of low concentrations of the denaturants (up-to 1M) hydrogen bonding network and van der Waals interaction play a role inducing changes in quaternary as well as tertiary structures without complete dissociation of the subunits. This is the first report of two large worm like, multi-domain proteins obeying twofold rule which is commonly found in small globular proteins. The free energy of stabilization (ΔG_u ^H ₂ ⁰) for the dimeric spectrin has been 20 kcal/mol lesser than the tetrameric from.     

Rotational freedom of tryptophan residues in proteins and peptides

We studied the rotational motions of tryptophan residues in proteins and peptides by measurement of steady-state fluorescence anisotropies under conditions of oxygen quenching. By fluorescence quenching we can shorten the fluorescence lifetime and thereby decrease the average time for rotational diffusion prior to fluorescence emission. This method allowed measurement of rotational correlation times ranging from 0.03 to 50 ns, when the unquenched fuorescence lifetimes are near 4 ns. A wide range of proteins and peptides were investigated with molecular weights ranging from 200 to 80 000. Many of the chosen substances possessed a single tryptophan residue to minimize the uncertainties arising from a heterogeneous population of fluorophores. In addition, we also studied a number of multi-tryptophan proteins. Proteins were studied at various temperatures, under conditions of self-association, and in the presence of denaturants. A wide variety of rotational correlation times were found. As examples we note that the single tryptophan residue of myelin basic protein was highly mobile relative to overall protein rotation whereas tryptophan residues in human serum albumin, RNase T1, aldolase, and horse liver alcohol dehydrogenase were found to be immobile relative to the protein matrix. These results indicate that one cannot generalize about the extent of segmental mobility of the tryptophan residues in proteins. This physical property of proteins is highly variable between proteins and probably between different regions of the same protein.     

Structure and dynamics of staphylococcal nuclease mutants as studied by fluorescence quenching techniques

Fluorescence techniques have been used to investigate the effect of mutations on the structure and dynamics of staphylococcal nuclease. An estimate of the accessibility to acrylamide of the enzyme's single tryptophan residue (Trp140) was obtained from the Stern-Volmer constant for fluorescence quenching. This was indicative of a partially buried tryptophan in the wild-type nuclease. Five single-site mutant nucleases (H124L, V66L, G88V, G79S and F76V) and one double mutant (V66L + G88V), with widely differing stabilities to denaturants, gave Stern-Volmer constants which were very similar to that of their parent enzyme. Studies of the temperature- and viscosity-dependence of quenching suggest that access by acrylamide to Trp140 is limited by diffusion rather than by protein structural fluctuations. Lifetime-resolved fluorescence anisotropy studies using steady-state instrumentation suggest that there is very little segmental motion of the Trp140; most of the anisotropy therefore decays due to protein rotation in the solution. Rotational correlation times for several nuclease mutants have been determined and these are very similar to that of the native nuclease. Thus it appears that these substitutions in the primary amino acid sequence, which have significant effects on the stability of the folded proteins, do not cause a significant change in the protein structure or dynamics around Trp140.     

Tryptophan fluorescence studies of ferredoxin:NADP reductase indicate the presence of tryptophan in or near the ferredoxin binding site

The tryptophan fluorescence properties of the flavoprotein ferredoxin:NADP reductase have been examined. Although not sensitive to changes in pH or salt concentration, the tryptophan fluorescence is affected by the presence of substrates for the flavoprotein. While NADP addition results in a slight quenching of the fluorescence, ferredoxin decreases the fluorescence by nearly 50%, suggesting the presence of tryptophan in or near the ferredoxin binding site. Titration of this effect gives a dissociation constant for the ferredoxin: flavoprotein complex which is similar to that obtained by spectral perturbations. This approach has also been used to demonstrate that a chemically modified ferredoxin which does not produce spectral perturbations when added to flavoprotein is capable of interacting with the flavoprotein although with a higher dissociation constant than for native ferredoxin.     

Erythroid spectrin in miceller detergents

We have studied the interaction of spectrin, the major protein of the erythrocyte cytoskeleton, with four commonly used detergents at concentrations above their critical miceller concentrations (cmc). Fluorescence spectroscopic studies on the emission intensity, steady state polarization, quenching with acrylamide, and time-resolved fluorescence measurements were done with spectrin in anionic detergents, e.g., SDS, deoxycholate, and nonionic detergents, e.g., Triton-X-100 and octylglucoside at concentrations double their respective cmc's. The spectrin-detergent complexes in all four systems have been characterized by far-UV CD and measurements on tryptophan fluorescence in combination with fluorescence of the extrinsic probe, pyrene. Tryptophan fluorescence studies revealed quaternary structural changes due to unzipping of the spectrin subunits in Triton-X-100 without complete dissociation. Both Triton-X-100 and SDS were found to partially denature spectrin indicated by the far-UV CD. Octylglucoside and deoxycholate are shown to have the least structural perturbations on the cytoskeletal protein, rationalizing the use of octylglucoside, in particular and also deoxycholate to be the most effective in preparing cytoskeletal fractions from erythrocytes rather than the Triton-X-100 that has long been used for preparing the Triton shells.     

Spectroscopic studies on the interaction of a water soluble porphyrin and two drug carrier proteins

The interaction of meso-tetrakis(p-sulfonatophenyl)porphyrin (TSPP) sodium salt to human serum albumin and beta-lactoglobulin was studied by steady-state and dynamic fluorescence at different pH of aqueous solutions. The formation of TSPP J-aggregates and a noncovalent TSPP-protein complex was monitored by fluorescence titrations, which depend on pH and on the protein nature and concentration. The complex between TSPP and protein displays a heterogeneous equilibrium with large changes in the binding strength versus pH. The large reduction of the effective binding constant from pH 2 to 7 suggests that electrostatic interactions are a major contribution to the binding of TSPP to the aforementioned proteins. TSPP aggregates and TSPP-protein complex exhibit circular dichroism induced by the presence of the protein. Circular dichroism spectra in the ultraviolet region show that the secondary structure of both proteins is not extensively affected by the TSPP presence. Protein-TSPP interaction was also examined by following the intrinsic fluorescence of the tryptophan residues of the proteins. Fluorescence quenching by acrylamide and TSPP itself also point to small changes on the protein tertiary structure and a critical distance R(0) approximately 56 A, between tryptophan and bound porphyrin, was estimated using the long distance F?rster-type energy transfer formalism.     

Large solutes induce structural perturbations in proteins and membranes.

Structural perturbations in biopolymers with hydrophobic interiors i.e. specific proteins and dimyristoylphosphatidylcholine (DMPC) vesicles were investigated as a function of solute concentrations in the medium. 1,6-diphenyl-1,3,5-hexatriene (DPH) was used as fluorescent probe. Response of DPH was comparable to that of intrinsic tryptophan in BSA in terms of steady state and time resolved fluorescence. The solutes induced a decrease in steady state anisotropy as well as rotational correlation time (computed from lifetime measurements) for DPH in both proteins and membranes. Enhanced access of the quencher potassium iodide to tryptophan in bovine serum albumin (BSA) and ovalbumin, and enhanced terbium leakage in DMPC vesicles induced by various solutes concomitant with decreased anisotropy/correlation time were consistent with structural perturbations of the nature of defects or voids in these polymers.     

Structure characterization of human serum proteins in solution and dry state.

The present report describes application of advanced analytical methods to establish correlation between changes in human serum proteins of patients with coronary atherosclerosis (protein metabolism) before and after moderate beer consumption. Intrinsic fluorescence, circular dichroism (CD), differential scanning calorimetry and hydrophobicity (So) were used to study human serum proteins. Globulin and albumin from human serum (HSG and HSA, respectively) were denatured with 8 m urea as the maximal concentration. The results obtained provided evidence of differences in their secondary and tertiary structures. The thermal denaturation of HSA and HSG expressed in temperature of denaturation (Td, degrees C), enthalpy (DeltaH, kcal/mol) and entropy (DeltaS kcal/mol K) showed qualitative changes in these protein fractions, which were characterized and compared with fluorescence and CD. Number of hydrogen bonds (n) ruptured during this process was calculated from these thermodynamic parameters and then used for determination of the degree of denaturation (%D). Unfolding of HSA and HSG fractions is a result of promoted interactions between exposed functional groups, which involve conformational changes of alpha-helix, beta-sheet and aperiodic structure. Here evidence is provided that the loosening of the human serum protein structure takes place primarily in various concentrations of urea before and after beer consumption (BC). Differences in the fluorescence behavior of the proteins are attributed to disruption of the structure of proteins by denaturants as well as by the change in their compactability as a result of ethanol consumption. In summary, thermal denaturation parameters, fluorescence, So and the content of secondary structure have shown that HSG is more stable fraction than HSA.     

Examination of substrate-induced conformational changes in the Na+/glucose cotransporter

Conformations of the Na+/glucose cotransporter were examined using tryptophan fluorescence and substrates to induce cotransporter conformational changes. Addition of Na+ but not K+ or TMA+ resulted in a saturable quenching of tryptophan fluorescence with a K0.5 for Na+ of 28 mM. In the presence of saturating Na+ concentrations, d-glucose but not l-glucose, fructose, or phlorizin resulted in a partial return of tryptophan fluorescence to approximately 70% of the substrate-free levels. This return of tryptophan fluorescence was a saturable function of d-glucose concentration with a K0.5 of 43 microM. The three conformations were compared with respect to their sensitivity to tryptophan quench reagents. Acrylamide quenching was unaffected by substrates. In contrast, I- quenching decreased 40% in the presence of Na+, while Cs+ quenching increased 64%. Addition of saturating d-glucose concentrations resulted in the return of I- quenching to 90% of the substrate-free values and reduced Cs+ quenching to substrate-free levels. In contrast, phlorizin did not mimic the effect of d-glucose on tryptophan fluorescence. These results are interpreted in terms of a second substrate-induced cotransporter conformational change which based on similar substrate specificities appears directly related to cotransporter-mediated Na+ and d-glucose transport.     

Structural changes enhance the activity of Chainia xylanase in low urea concentrations

Low concentrations of urea (1.2 M) stimulated the activity of endo-xylanase from Chainia by 30%. Subtle structural changes in the monomeric protein were reflected in the secondary and tertiary structure of the enzyme as monitored by fluorescence and circular dichroism. Changes in lambda(max) of emission, the fluorescence intensity and the Stern-Volmer quenching constants for acrylamide, measured in the presence of urea, indicated changes in the microenvironment of the Trp residues, suggesting alterations in tertiary structure. The ellipticity changes at 220 nm and Selcon analysis reflected changes in the content of beta-sheet while both the near- and far-UV CD spectra indicated alterations in the secondary and tertiary structure of the protein in presence of urea. The dissociation constant values (K(d)) show very little change in the affinity of the enzyme for the substrate while the k(cat) values suggest enhanced turnover of the substrate in presence of urea. We suggest that low urea concentrations perturb the conformational state of xylanase leading to an open and a more flexible structure, resulting in enhanced catalytic rates.     

A role of glycosyl moieties in the stabilization of bitter gourd (Momordica charantia) peroxidase

The possible role of carbohydrate moieties in the stabilization of proteins has been investigated by using bitter gourd peroxidase as a model system. A comparative study of glycosylated and non-glycosylated isoenzymes of bitter gourd peroxidase was performed at various temperatures, pH, water-miscible organic solvents, detergents and chaotropic agent like urea. The pH-optima and temperature-optima of both glycosylated and non-glycosylated isoforms of bitter gourd peroxidase remained unchanged. The probes employed were changes in the enzyme activity and fluorescence. The glycosylated form of peroxidase retained greater fraction of enzyme activity against the exposure caused by various physical and chemical denaturants. The unfolding of both forms of enzyme in the presence of high urea concentrations, studied by fluorescence, indicated greater perturbations in the conformation of non-glycosylated preparation. The different properties examined thus indicated that glycosylation plays an important role in the stabilization of native conformation of proteins against the inactivation caused by various types of denaturants.     

Structural stabilization of [2Fe-2S] ferredoxin from Halobacterium salinarum

The ferredoxin of the extreme haloarchaeon Halobacterium salinarum requires high (>2 M) concentration of salt for its stability. We have used a variety of spectroscopic probes for identifying the structural elements which necessitate the presence of high salt for its stability. Titration of either the fluorescence intensity of the tryptophan residues or the circular dichroism (CD) at 217 nm with salt has identified a structural form at low (<0.1 M) concentration of salt. This structural form (L) exhibits increased solvent exposure of W side chain(s) and decreased level of secondary structure compared to the native (N) protein at high concentrations of salt. The L-form, however, contains significantly higher levels of both secondary and tertiary structures compared to the form (U) found in highly denaturing conditions such as 8 M urea. The structural integrity of the L-form was highly pH dependent while that of N- or U-form was not. The pH dependence of either fluorescence intensity or CD of the L-form showed the presence of two apparent pK values: approximately 5 and approximately 10. The structural integrity of the L-form at low (<5) pH was very similar to that of the N-form. However, titration with denaturants showed that the low pH L-form is significantly less stable than the N-form. The increased destabilization of the L-form with the increase in pH was interpreted to be due to mutual Coulombic repulsion of carboxylate side chains (pK approximately 6) and due to the disruption of salt bridge(s) between ionized carboxylates and protonated amino groups (pK approximately 10). Estimation of solvent accessibility of W residues by fluorescence quenching, and measurement of decay kinetics of fluorescence intensity and anisotropy strongly support the above model. Polylysine interacted stoichiometrically with the L-form of ferredoxin resulting in nativelike structure. In conclusion, our studies show that high concentration of salt stabilizes the haloarchaeal ferredoxin in two ways: (i) neutralization of Coulombic repulsion among carboxyl groups of the acidic residues, and (ii) salting out of hydrophobic residues leading to their burial and stronger interaction.     

Interaction of dystrophin rod domain with membrane phospholipids. Evidence of a close proximity between tryptophan residues and lipids

Dystrophin is assumed to act via the central rod domain as a flexible linker between the amino-terminal actin binding domain and carboxyl-terminal proteins associated with the membrane. The rod domain is made up of 24 spectrin-like repeats and has been shown to modify the physical properties of lipid membranes. The nature of this association still remains unclear. Tryptophan residues tend to cluster at or near to the water-lipid interface of the membrane. To assess dystrophin rod domain-membrane interactions, tryptophan residues properties of two recombinant proteins of the rod domain were examined by (1)H NMR and fluorescence techniques in the presence of membrane lipids. F114 (residues 439-553) is a partly folded protein as inferred from (1)H NMR, tryptophan fluorescence emission intensity, and the excited state lifetime. By contrast, F125 (residues 439-564) is a folded compact protein. Tryptophan fluorescence quenching shows that both proteins are characterized by structural fluctuations with their tryptophan residues only slightly buried from the surface. In the presence of negatively charged small vesicles, the fluorescence characteristics of F125 change dramatically, indicating that tryptophan residues are in a more hydrophobic environment. Interestingly, these modifications are not observed with F114. Fluorescence quenching experiments confirm that tryptophan residues are shielded from the solvent in the complex F125 lipids by a close contact with lipids. The use of membrane-bound quenchers allowed us to conclude that dystrophin rod domain lies along the membrane surface and may be involved in a structural array comprising membrane and cytoskeletal proteins as well as membrane lipids.     

Inverse effects of D-galactosamine and inorganic phosphate on glycogenolysis in isolated rat hepatocytes.

Trichloroethanol is an efficient quencher of indole fluorescence of model compounds and proteins [Eftink, M. R. and Ghiron, C. A. (1976) J. Phys. Chem. 80, 486--493]. At low quencher concentrations, the quenching follows the classical Stern-Volmer law. Bimolecular rate constants calculated from measured quenching constants and lifetimes are equal to 6 X 10(9) M-1s-1 and 1.2 X 10(9) M-1s-1 for N-acetyltrypotophanamide and wheat germ agglutinin, respectively. Upon ultraviolet irradiation in the presence of trichloroethanol, transformation of fluorescent tryptophan occurs, leading to a fluorescent photoproduct. This can be easily used as a method for the quantitative determination of fluorescent tryptophan residues in proteins. In good agreement with previous results, two fluorescent tryptophan residues per polypeptide chain are found in wheat germ agglutinin. Concomitantly with the photochemical reactions, the hemagglutinating protein activity and its affinity constant towards chitin oligomers are reduced. A probable location of tryptophan residues in the binding sites of wheat germ agglutinin is proposed.     

Spectral studies of horse heart porphyrin cytochrome c

Removal of the heme iron from cytochrome c to generate porphyrin cytochrome c relieves the quenching of porphyrin fluorescence and enhances the fluorescence of the single tryptophan residue and the 4 tyrosine residues. The intensity of the porphyrin fluorescence is not perturbed by denaturation of the protein at neutral pH using either urea or guanidine hydrochloride. However, the amplitude of tryptophan fluorescence is increased by these denaturants from 5 to about 85% of a model tryptophan residue using solutions of 2 microM tryptophan. In contrast to cytochrome c, the tryptophan fluorescence amplitude of denatured porphyrin cytochrome c is independent of pH over the range pH 3.0 to 7.4. Acidification of solutions of either native or denatured porphyrin cytochrome c markedly alters both the visible absorbance and fluorescence of the protein consistent with protonation of two pyrrole nitrogens on the porphyrin. Preliminary analysis of the spectral changes occurring in the acid transition suggests the presence of an intermediate form having only one of these two pyrrole nitrogens protonated.     

pH-induced conformational states of bovine growth hormone

The folding behavior of bovine growth hormone (bGH) is examined by chemical and pH denaturation using several spectroscopic probes of protein secondary and tertiary structure. Partially denaturing concentrations of urea eliminate the native-state quenching of intrinsic tryptophan fluorescence, from the single protein tryptophan, but the fluorescence emission spectrum is not red-shifted like the unfolded state, and the protein retains substantial secondary structure. A neutral-to-acid pH shift also eliminates tryptophan quenching; however, the loss of quenching is not accompanied by an emission red-shift. In addition, the protein undergoes a pH-dependent UV absorbance transition; the changes in absorptivity have the same midpoint as the transition associated with the change in intrinsic tryptophan fluorescence. The magnitude of the absorption transition is similar to that observed previously for urea denaturation of the protein. In a similar fashion, a pH-dependent CD transition is also observed; however, the transition occurs at a higher pH. The behavior of the various optical probes indicates that the pH-induced conformational transition produces a highly populated species in which the microenvironment surrounding the single protein tryptophan residue resembles that observed during the urea-induced unfolding/refolding transition. The pH-induced changes in tertiary structure occur at a lower pH than the changes associated with a portion of the secondary structure. Proton NMR of the low-pH intermediate indicates that the three His and six Tyr resonances are indistinguishable from the unfolded state. The intermediate(s) observed by either chemical or pH-induced denaturation resemble(s) a molten globule state which contains significant secondary structure. The residual secondary structure present in the intermediate could be nonnative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectral characteristics of muscle aspartyl- and valyl-tRNA- synthetases and their complexes with substrates in normal conditions and after prolonged starvation

Spectral characteristics of aminoacyl-tRNA-synthetases (ARSases) isolated from muscles of normal rabbits and of those fasted for a long time were studied by the methods of fluorescence and differential spectroscopy. Fluorescence spectra and differential absorption spectra of the compared proteins evidenced for more hydrophobic surrounding of tryptophanyls and their less accessibility for Cs+ ions in proteins of fasted animals. Interaction of aspartyl- and valyl-tRNA-synthetases from muscles of normal and long-fasted rabbits with substrates is accompanied by the essential quenching of tryptophan fluorescence of ARSases. Equilibrium constants of substrate binding calculated from the fluorescence quenching curves are higher for specific amino acids than for non-specific ones. The effect of a long-wave shift of fluorescence spectra under marginal excitation of tryptophan residues was used to determine structural differences of enzymes in norm and under fasting and to find their structural peculiarities during formation of aminoacyl adenylate. Aminoacyl-tRNA-synthetases (ARSases) are key enzymes of the protein biosynthesis. High specificity of their interaction with substrates is the basis for the accuracy of genetic information implementation, namely translation of the genetic code. Molecular mechanisms of substrates "recognition" by ARSases are the objects of great attention of researchers.     

Detection, characterization, and quenching of the intrinsic fluorescence of bovine heart cytochrome c oxidase

The intrinsic fluorescence of lauryl maltoside solubilized bovine heart cytochrome c oxidase has been determined to arise from tryptophan residues of the oxidase complex. The magnitude of the fluorescence is approximately 34% of that from n-acetyltryptophanamide (NATA). This level of fluorescence is consistent with an average heme to tryptophan distance of 30 A. The majority of the fluorescent tryptophan residues are in a hydrophobic environment as indicated by the fluorescence emission maximum at 328 nm and the differing effectiveness of the quenching agents: Cs+, I-, and acrylamide. Cesium was ineffective up to a concentration of 0.7 M, whereas quenching by the other surface quenching agent, iodide, was complex. Below 0.2 M, KI was ineffective whereas between 0.2 and 0.7 M 15% of the tryptophan fluorescence was found to be accessible to iodide. This pattern indicates that protein structural changes were induced by iodide and may be related to the chaotropic character of KI. Acrylamide was moderately effective as a quenching agent of the oxidase fluorescence with a Stern-Volmer constant of 2 M-1 compared with acrylamide quenching of NATA and the water-soluble enzyme aldolase having Stern-Volmer constants of 12 M-1 and 0.3 M-1, respectively. There was no effect of cytochrome c on the tryptophan emission intensity from cytochrome c oxidase under conditions where the two proteins form a tight, 1:1 complex, implying that the tryptophan residues near the cytochrome c binding site are already quenched by energy transfer to the homes of the oxidase. The lauryl maltoside concentration used to solubilize the enzyme did not affect the fluorescence of NATA.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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.