Site-directed Mutagenesis of Either the Highly Conserved Trp-22 or the Moderately Conserved Trp-95 to a Large, Hydrophobic Residue Reduces the Thermodynamic Stability of a Spectrin Repeating Unit*

(Received for publication, January 24, 1997, and in revised form, May 24, 1997)

Dennis P. Pantazatos and Ruby I. MacDonald Dagger

From the Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

As reported previously (MacDonald, R. I., Musacchio, A., Holmgren, R. A., and Saraste, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1299-1303), an unfolded peptide was obtained by site-directed mutagenesis of Trp-22 to Ala in the cloned, wild type 17th repeating unit (alpha 17) of chicken brain alpha -spectrin. Trp occurs in position 22 of nearly all repeating units of spectrin. In the present study, Trp-22 was mutated to Phe or to Tyr to compare thermodynamic stabilities of urea-induced unfolding of alpha 16 and mutants thereof. alpha 16 was chosen for this study instead of alpha 17, because alpha 16 has two tryptophans, allowing urea-induced unfolding to be tracked by the fluorescence of the Trp remaining in each mutant peptide and by circular dichroism in the far UV.

The free energies of unfolding of W22Y and W22F were 50% that of alpha 16, showing that Trp-22 is crucial in stabilizing the triple helical bundle motif of the spectrin repeating unit. Mutation of the moderately conserved Trp-95 of alpha 16 to Val, which occupies position 95 in alpha 17, also yielded a peptide with 50% of the free energy of unfolding of alpha 16. Thus, the thermodynamic stability of a given spectrin repeating unit may depend on both moderately and highly conserved tryptophans. Different structural roles of Trp-22 and Trp-95 in alpha 16 are suggested by the slightly higher wavelength of maximum emission of Trp-22, the greater acrylamide quenching of Trp-95 than Trp-22, and the longer lifetime of Trp-95. For comparison with alpha 16, urea-induced unfolding of spectrin dimer isolated from human red cells was monitored by far UV-CD and by tryptophan fluorescence. Thermodynamic parameters could not be rigorously derived for the stability of spectrin dimer because unfolding of spectrin dimer involved more than two states, unlike unfolding of cloned repeating units. However, the similar midpoints of CD-monitored denaturation curves of alpha 16 and spectrin dimer, i.e. 2.7 and 3.2 M urea, respectively, indicate that investigation of cloned repeating units of spectrin can provide physiologically relevant information on these structures.


INTRODUCTION

The flexibility of the cytoskeletal protein spectrin is due to domains of 20 (alpha  subunit) and 17 (beta  subunit), linearly connected repeating units, each of which is folded into a triple-helical bundle (1, 2). It should be noted that the widely accepted term "repeating unit" does not imply sequence homology, since only one to two dozen amino acids are highly conserved among the 106-119 residues of these units of spectrin (3), as well as those of the closely related alpha -actinin (4) and dystrophin (5). Mutations of some of these conserved residues have pathologic consequences in organisms as diverse as Drosophila (6) and man (3, 7), although mutations of nonconserved residues of at least human erythroid spectrin have also been associated with certain hemolytic anemias (3, 7). These findings warrant further investigation of the relationship between the sequences of the repeating units and their biochemical and biophysical properties. A promising approach is the study of cloned repeating units that can be selectively mutated, as well as expressed singly or in numbers optimal for the measurement and analysis of those biochemical and biophysical properties. The feasibility of cloning one or more repeating units was first demonstrated by Winograd et al. (8) who obtained repeating units in a native-like conformation only when the spectrin cDNA coded for an integral number of repeats, i.e. when the cDNA was "conformationally phased."

Our interest is focussed on the tryptophan that is nearly invariant among the repeating units of spectrin (3), alpha -actinin (4) and dystrophin (5). In x-ray crystallography (9) and NMR (10) structures of two different triple helical, single repeating units of spectrin, Trp-22 is located in a site between the bulk phase and the region bounded by the three alpha  helices. This partially shielded position of Trp-22 is also indicated by its blue-shifted wavelength of maximum emission in alpha 17, the cloned 17th repeating unit of chicken brain alpha -spectrin (11). To probe the role of the highly conserved Trp-22 in the folding of the 17th repeating unit of chicken brain alpha -spectrin, alpha 17, we mutated Trp-22 to alanine and found that alpha 17 W22A was unfolded, suggesting that Trp-22 promotes stable folding of a repeating unit (11).

More conservative mutations of Trp-22 to Phe or to Tyr were made in the present study to obtain folded Trp-22 mutants for quantitative measurement of the importance of Trp-22 for the thermodynamic stability of a spectrin repeating unit. The 16th repeating unit (alpha 16) of chicken brain alpha -spectrin was chosen for the present study instead of alpha 17, because alpha 16 has two tryptophans instead of the single one in alpha 17. Hence, the fluorescence of the tryptophan remaining after site-directed mutagenesis of either the highly conserved Trp-22 or the less highly conserved Trp-95 could be measured to monitor tertiary structure during urea-induced unfolding of the mutant peptides. Secondary structure was monitored during urea-induced unfolding by CD at 222 nm. The free energies of unfolding of mutant peptides W22F and W22Y were 50% of alpha 16, establishing that Trp-22 significantly stabilizes the folding of alpha 16.

A major contribution to understanding how folding of the repeating unit is stabilized has been made by modeling the amino acid sequences of the helical regions of repeating units of spectrin and dystrophin (12). Modeling studies have revealed the arrangement of these residues in repeating heptad patterns designated "a" through "g," as depicted in Fig. 1 and indicated in Table I. The repeating heptad pattern is thought to stabilize each triple helical repeating unit, because the "a" and "d" residues, which are located in the inter-helical region, are frequently nonpolar so that one of these residues may form a hydrophobic association with a nonpolar a or d residue in an adjacent helix, whereas "e" and "g" residues, which are located between the inter-helical region and the bulk phase, are frequently charged so that one of these residues may form an inter-helical salt bridge with an e or g residue of opposite charge in an adjacent helix (12). As a large, hydrophobic residue in a g position, however, Trp-22 appears to be anomalous, in contrast with the less highly conserved Trp-95 which occurs in an a position. Together with the current finding that Trp-22 significantly increases the thermodynamic stability of alpha 16, the anomalous position of Trp-22 in the repeating heptad pattern suggests that Trp-22 may be involved in a novel mechanism of stabilizing repeating units of spectrin.


Fig. 1. Part of each of the three alpha  helices of a repeating unit of spectrin is shown in the form of a helical wheel which consists of a repeating heptad of amino acids, a-g. As found in spectrin and dystrophin (12), certain a and d residues are nonpolar so as to form hydrophobic interactions with an a or d residue on an adjacent alpha  helix, whereas certain e and g residues are charged so as to form salt bridges with an e or g residue of opposite charge on an adjacent alpha  helix.
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Table I. Amino acid sequences of the 16th repeating unit (alpha 16) of chicken brain alpha -spectrin and the peptides produced for this study

Hyphens indicate unchanged residues. The repeating heptad pattern, a through g, of each alpha -helix, which helps to stabilize the triple-helical bundle (12), is indicated as shown (9, 10).

1       10        20        30        40        50
*        *         *         *         *         *
       gabcdefgabcdefgabcdefgabcd        abcdefgabcdef
 alpha 16 AKLNESHRLHQFFRDMDDEESWIKEKKLLVSSEDYGRDLTGVQNLRKKHKRLEA
W22Y ---------------------Y--------------------------------
W22F ---------------------F--------------------------------
W22Y,S74L ---------------------Y--------------------------------
W22Y,V92M ---------------------Y--------------------------------
W95V ------------------------------------------------------
    60        70        80        90       100       110
     *         *         *         *         *         *
gabcdefgabcdefgabcdef     abcdefgabcdefgabcdefgabcd
 alpha 16 ELAAHEPAIQGVLDTGKKLSDDNTIGKEEIQQRLAQFVDHWKELKQLAAARGQRLE
W22Y --------------------------------------------------------
W22F --------------------------------------------------------
W22Y,S74L -------------------L------------------------------------
W22Y,V92M -------------------------------------M------------------
W95V ----------------------------------------V---------------


EXPERIMENTAL PROCEDURES

Materials

Chemicals

Sources of chemicals were as follows: ultrapure, enzyme grade urea, dithiothreitol and protein molecular weight standards for SDS-PAGE1 from Life Technologies, Inc.; D-10-camphorsulfonic acid from Aldrich; molecular biology grade acrylamide for Stern-Volmer quenching from BDH Laboratory Supplies, Poole, UK; ultrapure acrylamide for SDS-PAGE from Life Technologies Inc.; acrylamide for DNA sequencing gels ("Long Ranger") from AT Biochem; electrophoresis grade agarose for DNA gels from International Biotechnologies, Inc.; 35S-ATP from NEN Life Science Products; isopropyl beta -D-thiogalactoside from Research Organics, Inc.; Bacto-tryptone, yeast extract, and Bacto-agar from Difco; HindIII, BamHI, and T4 ligase from Promega; NCOI from New England Biolabs; AmpliTaq DNA polymerase and reagents for PCR from Perkin-Elmer; Sequenase II and reagents for DNA sequencing from U. S. Biochemical Corp.; phenol from Fluka Chemie, AG. Other chemicals were ultrapure, molecular biology or analytical reagent grade from Sigma. The chicken brain alpha -spectrin cDNA, some of the oligonucleotides, and some of the pET8c vector were generous gifts from Dr. Matti Saraste. The remaining oligonucleotides were synthesized by the Northwestern Biotechnology Facility or by Integrated DNA Technologies, Inc.

Methods

Preparation of Constructs

Constructs were prepared by standard methods (13, 14) as described previously (11). cDNA for the peptides was prepared by oligonucleotide-directed PCR of chicken brain alpha -spectrin cDNA (15) by AmpliTaq polymerase (Perkin-Elmer). Mutations W22Y, W22F, and W95V were introduced in oligonucleotides, as described previously (11). Amplified DNA fragments were isolated from agarose gels, ligated to pET8c vector (16), and used to transform BL21(DE3) strain Escherichia coli made competent by treatment with RbCl (17). Transformed cells were selected by growth on ampicillin plates. The sequences of inserted cDNAs of all plasmids were verified by DNA sequencing and are shown in Table I. The sequence of alpha 16 is as described previously (11), except that alanine is added at the N terminus which then is A and not K, and is exactly the same as the corresponding sequence from chicken brain alpha -spectrin (15).

Expression and Purification of Peptides

The peptides were obtained by growing plasmid-containing cells in 3 liters of Luria-Bertani medium + 100 µg/ml ampicillin at 37 °C until the A600 nm was between 0.2 and 0.6. Isopropyl beta -D-thiogalactoside was added to a concentration of 0.5 mM and growth continued for another 3 h. After pelleting in the JS-4.2 rotor of a Beckman J-6B centrifuge at 3,297 × g for 1 h and storage at -20 °C, the cells were sonicated in about 10 volumes of chilled, 0.02 M Tris-HCl, pH 8, + 1 mM EDTA + 0.15 mM phenylmethylsulfonyl fluoride + 1 mM dithiothreitol for 5, 1-min sonication periods alternating with 1-min rest periods. Cell debris was removed at 4 °C by ultracentrifugation in a type 45Ti rotor for 1 h at 186,000 × g or by centrifugation in a Sorvall SS34 rotor for 1 h at 27,000 × g. The supernatant was passed through a 0.45-µm filter and a 0.2-µm filter prior to DEAE-5PW high performance liquid chromatography (Toso-Haas). Peptides were detected on 15% SDS-PAGE gels in 3 M urea, further purified on Sephacryl S-100 (Sigma) and/or Q-Sepharose (Sigma) prior to desalting on 10DG columns (Bio-Rad) and stored at -20 °C in 10 mM sodium phosphate, pH 8. Peptides were at least 99% pure (with the exception of W22F which was at least 97% pure) by densitometry of SDS-PAGE gels. Yields were 10-100 mg per 3 liters of culture.

Peptide concentrations were determined from their absorbance at 280 nm, their tryptophan and tyrosine contents and the molar extinction coefficients at 280 nm for tryptophan and tyrosine (18). Exposure of the peptides to 6 M guanidine hydrochloride for comparison with native peptides (19) had a negligible (<5%, except for alpha 16 W22F at 8%) effect on their absorbance. The first 10 amino acids at the N terminus of the wild type, 16th repeating unit (alpha 16, formerly R16 (11)), were sequenced by Dr. Joseph Leykam at the Macromolecular Structure Facility, Michigan State University, and found to be the same as in Table I. Mass spectrometry analysis of all six peptides by Dr. Richard Milberg at the University of Illinois, Urbana, gave the expected Mr ± 0.1-0.2%.

According to published methods (20), a crude extract of spectrin was obtained by incubating red cell ghosts, which had been prepared from freshly drawn human red cells, in 0.5 mM beta -mercaptoethanol + 0.02 mM diisopropyl fluorophosphate + 0.1 mM EDTA, pH 9, at 37 °C. The crude extract was concentrated by ultrafiltration and layered on a Sepharose CL-4B column to isolate spectrin dimers. The spectrin dimer concentration was calculated from its extinction coefficient at 280 nm, i.e. 10.0 for a 1% solution (20). Densitometry of spectrin analyzed by SDS-PAGE on 8% polyacrylamide gels showed it to be at least 99% pure.

Fluorescence Measurements

8 or 10 M stock solutions of urea were made on the day of use by dissolving the urea in 10 mM sodium phosphate, pH 7.4, to maintain a pH of 8 in the peptide-containing samples. For Stern-Volmer measurements 1 M acrylamide stock solutions were also prepared on the day of use. All samples gave an absorbance at 295 nm of less than 0.1 in a cell of 1 cm path length so that correction for an inner filter effect was unnecessary (21). Steady-state fluorescence of 3 to 5 µM peptide or 50 µg/ml spectrin dimer in a cell of 0.5 cm path length was measured at room temperature with an AlphaScan fluorimeter (Photon Technology International) at a 4 or 6 nm band pass. Peptidyl tryptophan was excited at 295 nm and generally scanned from 300 to 400 nm. Raman and Rayleigh signals were subtracted from each scan. Measurements of urea-containing samples were performed at least 1 h after additions of the peptide to solutions at the appropriate urea concentrations. After scanning, the samples were usually stored at 4 °C overnight and re-scanned the next day to detect any changes in the readings that might alter the evaluation of thermodynamic parameters of unfolding. As no such changes were detected, all samples appeared to have attained equilibrium after an hour at room temperature. Fluorescence lifetimes were measured at room temperature in a LS100, nanosecond pulse fluorometer (Photon Technology International) with a hydrogen lamp in the time-correlated, single photon counting mode. Samples in a cell of 0.5 cm path length were excited at 297 nm and the emission recorded at 330 nm. To correct for the excitation pulse, light scattering of a second sample of 0.1 mg/ml glycogen was measured before each excitation of the peptide sample. Decays were fit to one or more exponentials by an iterative procedure based on the Marquardt algorithm (22). chi 2 values ranged from 0.9 to 1.2.

Circular Dichroism Measurements

Far-UV CD spectra of peptides at 5 µM in 10 mM sodium phosphate, pH 8, or of spectrin dimer at 50 µg/ml in 0.1 M NaCl + 0.01 M Tris-HCl, pH 7.5, + 0.1 mM EDTA + 0.1 mM beta -mercaptoethanol in a 0.1-cm quartz cell were taken at room temperature with a Jasco 500C spectropolarimeter. Calibration was performed with a solution of D-10-camphorsulfonic acid (23), and base lines were subtracted from all samples. CD measurements are reported as mean residue ellipticities, [theta ], in degrees·cm2/dmol. Spectra were scanned from 250 to 190 nm in 1-nm steps with a 4-s time constant at a rate of 20 nm/min. The photomultiplier voltages corresponding with the reported values were less than 500 V.

Calculation of m, U50%, and Delta GH2OUN

We followed the linear extrapolation method (24), as modified (25) to include nonlinear fitting of data to Equation 1 below, which has been evaluated recently with simulated fluorescence data (26). Application of this method has given the same thermodynamic values for unfolding (UN) of model peptides regardless of the mechanism assumed for solute-induced denaturation, i.e. denaturant binding or solvent transfer (27). This method has also yielded the same Delta G<SUP><UP>H<SUB>2</SUB>O</UP></SUP><SUB><UP>UN</UP></SUB> values of thioredoxin unfolding when induced either by thermal or by solute denaturation (28) and the same Delta G<SUP><UP>H<SUB>2</SUB>O</UP></SUP><SUB><UP>UN</UP></SUB> values of RNase unfolding whether induced by urea or by guanidine hydrochloride (29). Our data for the cloned peptides met the requirements for analysis by this method, since measurements were made at equilibrium and urea-induced unfolding of cloned repeating units was reversible (Figs. 2 and 4) and consisted of two states with neither detectable intermediates (Fig. 5) nor significant aggregation of the native state (Fig. 9). In the case of spectrin dimer, however, plotting of fluorescence intensity at a single wavelength did not yield a smooth urea denaturation curve (not shown), apparently due to nonsimultaneous unfolding of its many repeating units, as well as nonhomologous domains. However, calculation of an intensity-averaged emission wavelength, lambda , for spectrin dimer, according to Ref. 30, plotted versus urea concentration yielded a smooth curve.


Fig. 2. Tryptophan emission spectra of spectrin repeating units were recorded at 3 or 5 µM in 10 mM sodium phosphate, pH 8, but the fluorescence intensities of all peptides have been adjusted to represent 3 µM peptide to facilitate their comparison. The ordinate for alpha 16 is at the upper left; the ordinate for W22Y; W22F; W22Y,S74L and W22Y,V92M is at the upper right and lower left, and the ordinate for W95V is at the lower right. Samples were excited at 295 nm. Solid lines indicate native peptide, lines of dashes indicate peptide unfolded in 4-5 M urea, and lines of dash-dot-dashes indicate peptide unfolded in 4-5 M urea and diluted in 10 mM sodium phosphate, pH 8, to promote refolding.
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Fig. 4. Far UV-CD spectra of spectrin repeating units at 1.2-3.6 µM in 10 mM sodium phosphate, pH 8, either not exposed to urea (solid lines) or unfolded in 4 M urea and refolded by dilution in buffer (dashed lines). Both samples were passed over a gel filtration column to eliminate remaining urea in the case of the refolded peptide or to maintain the same peptide concentration in the case of the native peptide. The spectra of refolded W22Y; W22Y,V92M, and W95V were increased by 2,000-3,000 degrees·cm2·dmol-1 to make them distinguishable from the spectra of the native forms of those native peptides.
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Fig. 5. Urea denaturation curves of fraction unfolded peptide based on tryptophan fluorescence (open circle ) or far UV-CD (bullet ) data fit to the equation describing peptide unfolding as a two-state, reversible process at equilibrium as described under "Experimental Procedures."
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Fig. 9. Molecular sizes of spectrin repeating units indicated by 90° light scattering versus peptide concentration. Measurements in the upper panel were obtained at excitation and emission wavelengths of 300 nm and those in the lower panel were obtain at excitation and emission wavelengths of 400 nm. In each panel the dashed line with the higher slope denotes bovine serum albumin and the dashed line with the lower slope denotes RNase A.
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Equation 1 (25) was solved for m, which is the slope of the transition of denaturation, and U50%, which is the urea concentration at 50% denaturation, from fluorescence (F) or [theta ]222 nm values. The product of m and U50% is the free energy of unfolding in the absence of urea, Delta G<SUP><UP>H<SUB>2</SUB>O</UP></SUP><SUB><UP>UN</UP></SUB>.
F={(&agr;<SUB><UP>N</UP></SUB>+&bgr;<SUB><UP>N</UP></SUB><UP>U</UP>)+(&agr;<SUB><UP>D</UP></SUB>+&bgr;<SUB><UP>D</UP></SUB><UP>U</UP>) <UP>exp</UP>[(m<UP>U</UP>−m<UP>U</UP><SUB>50%</SUB>)/RT]}/ (Eq. 1)
{1+<UP>exp</UP>[(m<UP>U</UP>−m<UP>U</UP><SUB>50%</SUB>)/RT]}
alpha  is the y intercept of the native (N) or denatured (D) state, beta  is the slope of the native (N) or denatured (D) state, U is the urea concentration, R is the gas constant, 1.987 kcal·mol-1K-1, and T is the temperature in K. To superimpose fluorescence and CD denaturation curves in Figs. 5 and 8, the fluorescence intensities and [theta ]222 nm from each experiment were converted into values corresponding to fractions of unfolded peptide. Fluorescence intensities at 320 or 330 nm (F), [theta ]222 nm, or fraction unfolded values based on fluorescence or CD measurements from two or more experiments for each peptide were pooled and analyzed by nonlinear regression with SigmaPlot 5.0 (Jandel Scientific).


Fig. 8. Denaturation curves of fraction of spectrin dimer unfolded versus urea concentration at room temperature, calculated from the measured [theta ]222 (open circle ) and from the intensity-averaged emission wavelength, lambda  (bullet ). The dimer concentration is 50 µg/ml in 0.1 M NaCl + 10 mM Tris, pH 7.5, + 0.1 mM EDTA + 0.1 mM beta -mercaptoethanol.
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90° Light Scattering

To determine the molecular masses of peptides under the conditions of fluorescence and CD measurement, peptides at 0.05 to 0.5 mg/ml in 10 mM sodium phosphate, pH 8, were excited at 300 or 400 nm in a cell of 0.5 cm path length, whereas light scattering was recorded at 300 or 400 nm and a band pass of 6 or 4 nm, respectively, in an AlphaScan fluorimeter. Standards were bovine serum albumin (Mr 66,000) and pancreatic RNase A (Mr 13,700), the diameters of which are small enough compared with the wavelength of the exciting light that only molecular size, but not shape, affect the signal (31). The least squares method was used to fit light scattering versus peptide concentration to a curve, the slope of which gave the molecular mass of the peptide by interpolation between the standards by the method of Lagrange (32).


RESULTS

Sequences of Constructs

Amino acid sequences of the six peptides cloned for this study are given in Table I. The peptides are 1) the 16th repeating unit (alpha 16) of chicken brain alpha -spectrin, 2) the W22Y mutation of alpha 16, 3) the W22F mutation of alpha 16, 4) W22Y with an artifactual second mutation, S74L, 5) W22Y with another artifactual second mutation, V92M, and 6) the W95V mutation of alpha 16. The peptides mutated at Trp-22 were cloned to assess its role in the stable folding of alpha 16, and W95V was cloned to compare the fluorescence properties of Trp-22 and Trp-95 and to probe the role of Trp-95 in the stable folding of alpha 16.

Tryptophan Fluorescence Spectra Indicate Reversible Unfolding of All Peptides in Urea

Fig. 2 contains the tryptophan emission spectra of the peptides listed in Table I. To obtain the fluorescence data, 2 aliquots of each peptide were incubated at room temperature in 4 (W22F and alpha 16) or 5 M urea (W22Y; W22Y,S74L; W22Y,V92M; W95V) for at least 1 h, after which each sample was diluted 10-fold with 4 or 5 M urea (Fig. 2, dashed line) or with buffer alone (Fig. 2, dash-dot-dashed line). As a control, a 3rd aliquot was incubated simultaneously in buffer alone and subsequently diluted 10-fold with the same buffer (solid line). The reversibility of unfolding of all peptides is shown by the coincidence of scans of native peptides with scans of re-folded peptides. In addition, the more intense fluorescence of Trp-95 in peptides with mutations of Trp-22 compared with the quenched fluorescence of Trp-22 in the peptide with mutated Trp-95 is striking, particularly since the emission maxima of these peptides are similar. Trp-95 in peptides mutated at Trp-22 to Phe or Tyr has a lambda max at 330 nm indicating its shielding from the bulk phase. Trp-22 in W95V is slightly less shielded than Trp-95 with a lambda max of 333 ± 0.0 nm in folded W95V and 334.8 ± 2.4 nm in refolded W95V. The lambda max of all unfolded peptides is red-shifted to 354 nm, indicating exposure of tryptophan(s) to the bulk phase.

Trp-22 Exhibits a Lower KSV of Acrylamide Quenching Than Trp-95

Acrylamide quenching of native peptides was performed to determine the accessibilities of Trp-22 and Trp-95 to the bulk phase. Differential quenching might explain the more intense fluorescence of Trp-95 in W22Y, W22F, W22Y,S74L, and W22Y,V92M than of Trp-22 in W95V (Fig. 2). The Stern-Volmer plots in Fig. 3 show that peptides with only Trp-95 gave a slope or Stern-Volmer constant, KSV, which is about 2.9 times that of the peptide with only Trp-22. Since KSV is the product of the fluorescence lifetime, tau , and the collisional quenching constant, kq (33), however, this difference could be due to a difference in either tau  and/or kq. Here the higher KSV of Trp-95 is due almost entirely to the longer average lifetime tau  of Trp-95 (3.4 ns) than Trp-22 (1.0 ns) and negligibly to a higher collisional quenching constant kq. These averages were calculated from lifetime distributions with the best fits to the decay of Trp-95 fluorescence, i.e. 4.21 ns (A = 0.67) + 1.73 ns (A = 0.33), and to the decay of Trp-22 fluorescence, i.e. 0.6 ns (A = 0.83) + 2.36 ns (A = 0.13) + 5.4 ns (A = 0.04). "A" denotes the fraction of the population with the stated lifetime.


Fig. 3. Stern-Volmer plots of tryptophan fluorescence in the absence of acrylamide (F0)/fluorescence in the presence of acrylamide (F) of W22F (down-triangle), W22Y (open circle ), W22Y,S74L (square ), W22Y,V92M (triangle ), and W95V (black-diamond ), each at 3 µM. Bars indicate standard errors.
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Reversibility of Urea Denaturation of All Peptides Is Also Shown by CD Data

CD spectra with about twice the positive signal at 190 nm as negative signal at 222 nm in Fig. 4 are typical of peptides with largely alpha -helical structure (34). The spectrum of each peptide in its native form (Fig. 4, solid line), furthermore, overlaps that of the same peptide which had been denatured in 4 M urea and subsequently refolded by dilution and removal of urea on a desalting column (Fig. 4, dashed line). This correspondence of the CD spectra of the untreated (solid line) and urea-treated but refolded (dashed line) forms of each peptide in Fig. 4 corroborates the fluorescence-monitored reversibility of urea denaturation in Fig. 2. Based on the mean residue ellipticity of a peptide with 100% alpha -helical structure, i.e. 36,000 degrees·cm2·dmol-1 at 222 nm (34), the % alpha -helicity of each peptide was calculated to be 56.5% for alpha 16, 28.4% for W22Y, 39.9% for W22F, 47.6% for W22Y,S74L, 40.4% for W22Y,V92M and 25.3% for W95V. The 56.6% value for alpha 16 is within the range of values obtained for alpha 16, averaging 66.7% ± 12.9. The helical content of each peptide is roughly commensurate with its free energy of peptide unfolding given in Fig. 6.


Fig. 6. Bar graph of thermodynamic parameters derived from the urea denaturation curves in Fig. 5. The first bar in each panel represents a fluorescence value and the second bar represents a CD value, each with its standard error. The upper row gives values for Delta G<SUP><UP>H<SUB>2</SUB>O</UP></SUP><SUB><UP>UN</UP></SUB> in kcal ·mol-1; the middle row gives values for U50% in M, and the lowest row gives values for m in kcal·mol-1·M-1.
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Urea Denaturation Curves Based on Fluorescence and CD Data Are Superimposable for All Peptides

Given that the conditions for thermodynamic analysis of the stability of peptide folding were satisfied, peptide unfolding was monitored at increasing concentrations of urea. Tryptophan fluorescence and ellipticity at 222 nm of urea-treated peptides were converted into fractions of unfolded peptide, plotted versus urea concentration, and fit to Equation 1 under "Experimental Procedures" describing peptide unfolding as a two-state, reversible process at equilibrium. In Fig. 5 urea denaturation curves based on tryptophan fluorescence (Fig. 5, open circles) coincide with urea denaturation curves based on the mean residue ellipticity at 222 nm (Fig. 5, solid circles) for all peptides with the possible exception of W22Y,S74L. The thermodynamic parameters from fluorescence and CD data in Fig. 6, however, are identical within standard error for all peptides, so that unfolding appears not to involve intermediate states (35).

Thermodynamic Parameters of Peptide Unfolding

Values for thermodynamic parameters from data in Fig. 5 are given in Fig. 6. The U50% representing the average of fluorescence and CD values range from 2.44 M urea for W22Y,S74L, 2.38 M urea for alpha 16, 1.77 M urea for W22Y,V92M, 1.38 M urea for W22Y, 1.37 M urea for W22F to 1.04 M urea for W95V. In contrast with these U50% values, the cooperativity of unfolding, m, ± S.E. is about the same for all peptides, with the possible exception of W22Y. Hence, the product of U50% and m, Delta G<SUP><UP>H<SUB>2</SUB>O</UP></SUP><SUB><UP>UN</UP></SUB>, ranges nearly in parallel with the U50% values from 4.5 kcal/mol for alpha 16, 4.3 kcal/mol for W22Y,S74L, 2.9 kcal/mol for W22Y,V92M, 2.3 kcal/mol for W22F, 2.0 kcal/mol for W22Y to 1.9 kcal/mol for W95V. Thus, at 25 °C and in 10 mM sodium phosphate, pH 8, the artifactual, second mutation in W22Y,V92M partially restores and in W22Y,S74L completely restores the significantly diminished stability of folding due to substitution of the highly conserved tryptophan Trp-22 with tyrosine.

Urea Denaturation Curves of Wild Type and W22Y,S74L Under More Physiological Conditions

We next examined the similarly stable alpha 16 and W22Y,S74L under more physiological conditions of ionic strength and temperature to assess whether a second mutation like S74L might compensate for the destabilizing effect of a mutation like W22Y in vivo and to enable comparison with the unfolding of intact spectrin dimer. Data in Fig. 7A were obtained at 25 °C and data in Fig. 7B were obtained at 37 °C, both in 0.14 M KCl + 10 mM sodium phosphate, pH 7.5. From their Delta G<SUP><UP>H<SUB>2</SUB>O</UP></SUP><SUB><UP>UN</UP></SUB> values in Table II, W22Y,S74L and the wild type are still equally stably folded at 25 °C and more stably folded than W22F, but the wild type is marginally more stably folded than W22Y,S74L at 37 °C.


Fig. 7. A, unfolding at 25 °C of 3 µM alpha 16 (black-down-triangle ), W22F (square ), and W22Y,S74L (open circle ) in urea + 0.14 M KCl + 10 mM NaP04, pH 7.5. B, unfolding at 37 °C of 3 µM alpha 16 (black-down-triangle ) and W22Y,S74L (open circle ) in urea + 0.14 M KCl + 10 mM sodium phosphate, pH 7.5. Fluorescence data were converted into fractions of unfolded peptide and fit to the equation describing peptide unfolding as a two-state, reversible process at equilibrium as described under "Experimental Procedures." The thermodynamic parameters are given in Table II.
[View Larger Version of this Image (23K GIF file)]

Table II. Free energies of peptide unfolding induced by urea in 0.14 M KCl at pH 7.5 and monitored by tryptophan fluorescence


Peptide m U50%  Delta GUN

kcal/mol · M M kcal/mol
 alpha 16 (25 °C)a 1.75  ± 0.19 2.73  ± 0.05 4.77  ± 0.52
W22F (25 °C) 1.92  ± 0.15 1.91  ± 0.04 3.66  ± 0.35
W22Y,S74L (25 °C) 1.61  ± 0.24 2.81  ± 0.07 4.54  ± 0.67
 alpha 16 (37 °C) 1.58  ± 0.21 1.89  ± 0.09 2.99  ± 0.49
W22Y,S74L (37 °C) 1.38  ± 0.09 1.50  ± 0.07 2.07  ± 0.23

a Temperature at which the measurement was made is given in parentheses.

Urea Denaturation of Spectrin Dimer Isolated from Human Red Cells

For comparison with urea-induced unfolding of alpha 16 in Fig. 7A, spectrin dimer was isolated from human red cells and its urea-induced denaturation followed by [theta ]222 and by tryptophan fluorescence in 0.1 M NaCl + 10 mM Tris-HCl, pH 7.5, + 0.1 mM EDTA + 0.1 mM beta -mercaptoethanol. Nearly identical with the average 66.7% alpha -helical content of alpha 16, the alpha -helical content of intact spectrin dimer was 68.2%, based on a 100% value of 36,000 degrees·cm2·dmol-1 (34). Both of the fraction unfolded curves in Fig. 8, one based on [theta ]222 and the other on the intensity-averaged emission wavelength, appear to describe a two-state transition of spectrin dimer unfolding. However, calculation of a putative [U]50% yielded 3.2 ± 0.69 M urea for the CD curve and 4.4 ± 0.68 M urea for the fluorescence curve, and calculation of a putative m yielded 0.29 ± 0.16 kcal/mol·M for the CD curve and 0.38 ± 0.12 kcal/mol·M for the fluorescence curve. Comparison of these values for spectrin dimer with those for the cloned peptides in Table II and Fig. 6 reveals the standard errors of these values for spectrin dimer to be about an order of magnitude larger than for the well-behaved [U]50% and m of the cloned peptides. Also, the 1.2 M difference in [U]50% from CD versus fluorescence data of intact spectrin dimer is much greater than the <0.2 M difference in [U]50% from CD versus fluorescence data of the cloned alpha 16 and mutant peptides. Finally, the values for m of spectrin dimer are very low, about 1/6 that of m for the cloned peptides. Thus, in contrast with the authentically two-state denaturation curves of cloned peptides in Fig. 5, the resemblance to a two-state transition of denaturation curves of spectrin dimer in Fig. 8 is misleading.

All Peptides Except for W22F Are Monomers at the Outset of Urea-induced Unfolding, According to 90° Light Scattering

Fig. 9 is a plot of 90° light scattering versus increasing concentrations of all six peptides to determine their molecular masses in solution. Data in the upper 3 panels were obtained at a band pass of 6 nm with the excitation and emission monochromators set at 300 nm, whereas data in the lower 3 panels were obtained at a band pass of 4 nm with the excitation and emission monochromators set at 400 nm. Standards are represented in each panel by the upper dashed line for bovine serum albumin (Mr 66,000) shown without data points and the lower dashed line for RNase A (Mr 13, 700) also shown without data points. The following masses ± S.E. were determined: 13,770 ± 1,628 for alpha 16; 19,244 ± 1,922 for W22F; 12,927 ± 1,098 for W22Y,S74L; 11,681 ± 253 for W22Y; 12,315 ± 1,114 for W22Y,V92M, and 14,040 ± 240 for W95V. As expected, these molecular masses obtained by light scattering are more variable and less accurate than those obtained by mass spectrometry (see "Experimental Procedures") but were necessary to assess the degree of aggregation of the peptides under the conditions of the fluorescence and CD measurements. alpha 16 has also been shown by Pascual et al. (10) to be a monomer by analytical ultracentrifugation, unlike the wild type, 14th repeating unit of Drosophila alpha -spectrin (36).

Although the W22F mutant peptide was not monomeric in its native state, its CD and fluorescence-based denaturation curves in Fig. 5 appear well-behaved in terms of 1) the coincidence of these curves, 2) the identity of thermodynamic parameters (± S.E.) obtained from these curves, and 3) the general appearance of these curves indicating that W22F unfolding is a two-state process. Thus, we have established that thermodynamic analysis of urea-induced unfolding of cloned repeating units is valid since peptide unfolding was reversible (Figs. 2 and 4), data were taken at equilibrium, unfolding involves two states from the sigmoidal and superimposable far UV-CD and tryptophan fluorescence denaturation curves (Fig. 5), and all peptides with the exception of W22F were monomers by 90° light scattering (Fig. 9).


DISCUSSION

Conservative Mutation of the Nearly Invariant Trp-22 Significantly Reduces the Thermodynamic Stability of Folding of a Repeating Unit of Spectrin

Mutation of the nearly invariant Trp-22 to tyrosine or to phenylalanine, which are most frequently substituted for tryptophan in nature (37), reduces the Delta G<SUP><UP>H<SUB>2</SUB>O</UP></SUP><SUB><UP>UN</UP></SUB> of alpha 16 of chicken brain alpha -spectrin by 50% (Fig. 6). Hence, even amino acids most closely resembling tryptophan in size and hydrophobicity fail to substitute for Trp-22 in maintaining the stability of folding of a wild type repeating unit of spectrin. As noted in the Introduction and indicated in Table I, Trp-22 does not occupy an a or d position in the repeating heptad pattern (Fig. 1), where stabilization of the triple helical bundle of the repeating unit may occur through hydrophobic interactions (12). Contrary to expectations for a large, hydrophobic amino acid, Trp-22 occupies a g position (Table I), where stabilization may occur through the formation of salt bridges (12). Furthermore, Trp-22 is unique in chicken brain alpha -spectrin as a large, hydrophobic amino acid which is highly conserved in a g position of the repeating heptad pattern (10, 15). Thus, the situation of the nearly invariant Trp-22 suggests a third type of mechanism for stabilizing the triple helical bundle motif of a repeating unit of spectrin, in addition to interhelical hydrophobic interactions and salt bridges (12).

Mutations of a Moderately Conserved and Two Nonconserved Residues Also Affect the Free Energy of Unfolding of alpha 16

It seemed possible that valine would be an adequate substitute for Trp-95 in alpha 16, since valine occupies position 95 of alpha 17 of chicken brain alpha -spectrin (15). On the other hand, as indicated in Table I, Trp-95 occurs in the a position of the repeating heptad pattern (9, 10) where hydrophobic interactions could impose stringent packing requirements (12). The validity of the latter point is supported by the 50% lower Delta G<SUP><UP>H<SUB>2</SUB>O</UP></SUP><SUB><UP>UN</UP></SUB> of W95V than alpha 16. In the crystal structure of the 14th repeating unit of Drosophila alpha -spectrin (9) hydrogen bonding of Trp-95 and Trp-22 to other residues appears unlikely. Therefore, destabilization resulting from substitution of either Trp-22 or Trp-95, particularly Trp-95 since it occupies an a position in the repeating heptad pattern, may be due to the creation of a cavity left by replacement of the tryptophan with a smaller amino acid. Also possibly indicating the importance of nonconserved amino acids among repeating units for their stability of folding, additional mutations in W22Y of S74L or of V92M, artifactually occurring during PCR, restored the Delta G<SUP><UP>H<SUB>2</SUB>O</UP></SUP><SUB><UP>UN</UP></SUB> of W22Y almost entirely or by about 50%, respectively. The greater stability of W22Y,V92M than W22Y can be ascribed to Met providing a more polar interaction than Val at position 92 which is an e position (Table I). In contrast, the Ser right-arrow Leu change cannot be rationalized in the same way, since position 74 is also an e position (Table I), which should be less well filled by substituting a polar residue like Ser with a more nonpolar residue such as Leu. It remains to be determined how S74L compensates for the destabilizing effect of W22Y. Whereas it would have been predicted that any substitution of a highly conserved residue such as the nearly invariant Trp-22 should result in destabilization of folding of a cloned repeating unit, it is less certain that mutations of moderately or even nonconserved residues in general would affect the thermodynamic stability of a repeating unit. This question may be particularly relevant to understanding the evolution of spectrin, e.g. by duplication of groups of repeating units (38), and the mechanism underlying hemolytic anemias correlated with single mutations of nonconserved amino acids (3, 7) and deserves further investigation.

Comparison of Urea-induced Denaturation of alpha 16 and W22Y,S74L under More Physiological Conditions

We compared urea-induced unfolding of W22Y,S74L and alpha 16, which exhibited similar Delta G<SUP><UP>H<SUB>2</SUB>O</UP></SUP><SUB><UP>UN</UP></SUB> values, in 0.14 M KCl + 10 mM sodium phosphate, pH 7.5, at 25 and 37 °C to assess the ability of S74L to compensate for W22Y under more physiological conditions. As discussed below, these data for alpha 16 were also necessary for comparison with data on the unfolding of spectrin dimer isolated from human red cells (Fig. 8). W22Y,S74L remained as stable as the wild type at 25 °C (Fig. 7A) and was only marginally less stable than the wild type at 37 °C (Fig. 7B). Despite the ability of mutations like S74L to compensate for the destabilizing mutation of Trp-22 in a cloned repeating unit, such secondary mutations are not found in vivo since Trp-22 is so highly conserved among repeating units of spectrin. It may be that the probability of secondary mutations like S74L occurring simultaneously in the same repeating unit as the destabilizing mutation of Trp-22 is extremely low in vivo and/or that Trp-22 is uniquely important in vivo for a second vital function, in addition to stable folding, for which a second mutation cannot compensate.

Comparison of Urea-induced Unfolding of alpha 16 with That of Human Red Cell Spectrin Dimers

Although the CD and fluorescence-based, urea denaturation curves of spectrin dimers in Fig. 8 resemble two-state transitions, the following observations indicate that more than two states are involved: 1) the order of magnitude greater standard errors of the putative [U]50% and m of the unfolding of human red cell spectrin dimer than those of the corresponding parameters of alpha 16 unfolding; 2) the much larger difference between the putative [U]50% of fluorescence and CD curves for spectrin dimer in Fig. 8 than for the definitive [U]50% of fluorescence and CD curves for cloned repeating units in Fig. 5, i.e. 1.2 M versus < 0.2 M, respectively; and 3) the much smaller m of spectrin dimer unfolding compared with the m of alpha 16 unfolding, 0.3 and 0.38 kcal/mol·M versus 1.75 kcal/mol·M, respectively. These differences also suggest that the multiple domains of spectrin dimer unfold independently of each other so that the midpoint of the dimer curve may represent the urea concentration at which half of the repeating units, instead of half of each repeating unit, have unfolded. Hence, thermodynamic parameters of unfolding of spectrin dimer were not obtained with the same rigor as thermodynamic parameters of unfolding of cloned, repeating units. Nevertheless, the roughly similar [U]50% values calculated from CD-monitored denaturation of alpha 16 and of human red cell spectrin dimer, 2.7 versus 3.4 M, respectively, indicate that the conformation of cloned alpha 16 could reasonably represent its conformation in a spectrin dimer. (alpha -Spectrin of chicken brain is identical to alpha -spectrin of avian red cells, since avian alpha -spectrin occurs as a single isoform (39). Thus, it is reasonable to compare alpha 16 of chicken brain alpha -spectrin with intact spectrin dimer from human red cells, which is more readily obtained than that from chicken red cells.)

The 4.77 ± 0.52 kcal/mol Delta G<SUP><UP>H<SUB>2</SUB>O</UP></SUP><SUB><UP>UN</UP></SUB> of unfolding of alpha 16 indicates a greater stability than the 3.4 kcal/mol first reported for a cloned repeating unit of dystrophin, measured by CD of heat or urea-denaturated peptide (40). A subsequent report by the same group (41) gave a significantly higher Delta G<SUP><UP>H<SUB>2</SUB>O</UP></SUP><SUB><UP>UN</UP></SUB> of 6.4 kcal/mol but about the same 72% alpha -helical content for the same repeating unit which had been extended at the C terminus by four residues. Differences in the consensus sequences of spectrin and dystrophin repeating units indicate that their folds may vary significantly despite their origin from a common motif (12, 42). Hence, comment on the reason for the differences between the free energy of unfolding of a spectrin repeating unit reported here and those found for the dystrophin repeating units (40, 41) would be premature. Since the original submission of this manuscript, two reports of similar differences in the unfolding of cloned, repeating units of red cell alpha -spectrin have come to our attention (43, 44). Further investigation is required to ascertain the structural significance of these differences.

Structural Basis of Differences in Fluorescence Intensities of Trp-22 and Trp-95

Multiple tryptophans in a protein frequently exhibit different quantum yields which change on protein unfolding, e.g. Ref. 30. In the present instance, the structural basis of (i) enhanced fluorescence of Trp-95 in W22F and W22Y, (ii) quenched fluorescence of Trp-22 in W95V, but (iii) quenched fluorescence of both Trp-95 and Trp-22 in alpha 16 (all relative to fluorescence in the unfolded peptides) is apparent in Fig. 10. Suggested by the structures of Yan et al. (9) and of Pascual et al. (10), Fig. 10 shows that both Trp-22 and Trp-95 lie near histidine 59, a strong tryptophan quencher (45). Pascual et al. (10) have detected nuclear Overhauser effects both between Trp-22 and His-59 and between Trp-95 and His-59. The quenching by histidine 59 of Trp-22 alone but not Trp-95 alone may be due to the different orientations of the two tryptophans to His-59, the smaller of the indole rings of Trp-22 but the larger of the indole rings of Trp-95 being closer to the histidine. The quenching of Trp-95 only when Trp-22 is also present, i.e. in the wild type, probably results from energy transfer from Trp-95 to Trp-22 which is within energy transfer distance. Quenching by histidine of Trp-22, but not Trp-95, in single tryptophan peptides also explains the lower KSV of Trp-22 than of Trp-95 (Fig. 3), since quenching by histidine is expected to shorten the lifetime of Trp-22 alone but not Trp-95 alone. This explanation is compatible with each tryptophan being exposed to the bulk phase to a similar but not identical degree, as affirmed by their wavelengths of maximum emission, i.e. 330 nm for W95V and 334 nm for Trp-22 (Fig. 2).


Fig. 10. Based on the x-ray crystal structure of Yan et al. (9) and the NMR structure of Pascual et al. (10), this ribbon diagram of alpha 16 as a triple-helical bundle constituting a single repeating unit indicates the proximity of Trp-22 and Trp-95 to His-59, as well as the sites of Ser-74 and Val-92 which were mutated unintentionally during PCR of W22Y,S74L and W22Y,V92M, respectively. The probable loop between helix B and helix C has been omitted since the crystal structure depicts helix B and helix C as a single, continuous alpha  helix (9).
[View Larger Version of this Image (34K GIF file)]

In summary, the importance of Trp-22 for the thermodynamic stability of alpha 16 and the similarity of the urea concentrations inducing denaturation of 50% of alpha 16 and intact spectrin constitute key evidence that cloned repeating units of spectrin are reasonable models of these structures as they function within cytoskeletal networks. We expect the recombinant DNA approach to continue to further our understanding of how triple-helical repeating units of spectrin are stabilized without mitigating the flexibility of this ubiquitous cytoskeletal protein.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant 1PO1 HL45168.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed.
1   The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; PCR, polymerase-chain reaction; SV, Stern-Volmer.

ACKNOWLEDGEMENTS

We thank Dr. Matti Saraste for the generous gift of chicken brain alpha -spectrin cDNA, pET8c vector, and primers and, together with Dr. Andrea Musacchio, for introducing us to this system; Dr. Robert Holmgren for advice on cloning; Drs. Theodore Jardetzky and Alfonso Mondragón for guidance in interpreting the crystal structure of Yan et al. (9); Dr. Alfonso Mondragón for providing the ribbon structure in Fig. 10; Steven Abel, Anupama Amaran, Jennifer Dasta, Tracy Irwin, Jennifer Liu, Sri Namperumal, and Wei-ming Su for technical assistance; Dr. Paul Loach for use of CD spectrometer; Dr. Francis Neuhaus for use of equipment; and Dr. Robert MacDonald for reading the manuscript.


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