(Received for publication, January 24, 1997, and in revised form, May 24, 1997)
From the Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208
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 (17) of chicken brain
-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
16
and mutants thereof.
16 was chosen for this study instead of
17,
because
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
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
16 to Val, which occupies position 95 in
17,
also yielded a peptide with 50% of the free energy of unfolding of
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
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
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
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.
The flexibility of the cytoskeletal protein spectrin is due to
domains of 20 ( subunit) and 17 (
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
-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), -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
helices. This partially shielded position of
Trp-22 is also indicated by its blue-shifted wavelength of maximum
emission in
17, the cloned 17th repeating unit of chicken brain
-spectrin (11). To probe the role of the highly conserved Trp-22 in
the folding of the 17th repeating unit of chicken brain
-spectrin,
17, we mutated Trp-22 to alanine and found that
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 (16) of
chicken brain
-spectrin was chosen for the present study instead of
17, because
16 has two tryptophans instead of the single one in
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
16, establishing that Trp-22 significantly
stabilizes the folding of
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 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.
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Materials
ChemicalsSources 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 -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
-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 ConstructsConstructs were prepared by
standard methods (13, 14) as described previously (11). cDNA for
the peptides was prepared by oligonucleotide-directed PCR of chicken
brain -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
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
-spectrin (15).
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 -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 16
W22F at 8%) effect on their absorbance. The first 10 amino acids at
the N terminus of the wild type, 16th repeating unit (
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
-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.
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). 2 values ranged
from 0.9 to 1.2.
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 -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, [
], 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.
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
values of
thioredoxin unfolding when induced either by thermal or by solute
denaturation (28) and the same
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,
, for spectrin dimer,
according to Ref. 30, plotted versus urea concentration
yielded a smooth curve.
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
[]222 nm values. The product of m
and U50% is the free energy of unfolding in the
absence of urea,
.
![]() |
(Eq. 1) |
![]() |
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).
Amino acid sequences of the six
peptides cloned for this study are given in Table
I. The peptides are 1) the 16th repeating unit (16) of chicken brain
-spectrin, 2) the W22Y mutation of
16, 3) the W22F mutation of
16, 4) W22Y with an artifactual second mutation, S74L, 5) W22Y with another artifactual second mutation, V92M, and 6) the W95V mutation of
16. The peptides mutated
at Trp-22 were cloned to assess its role in the stable folding of
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
16.
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 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
max at 330 nm indicating its shielding from the bulk
phase. Trp-22 in W95V is slightly less shielded than Trp-95 with a
max of 333 ± 0.0 nm in folded W95V and 334.8 ± 2.4 nm in refolded W95V. The
max of all unfolded
peptides is red-shifted to 354 nm, indicating exposure of tryptophan(s)
to the bulk phase.
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,
, and the collisional quenching constant, kq
(33), however, this difference could be due to a difference in either
and/or kq. Here the higher KSV of Trp-95 is due almost entirely to the
longer average lifetime
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.
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 -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%
-helical structure,
i.e. 36,000 degrees·cm2·dmol
1
at 222 nm (34), the %
-helicity of each peptide was calculated to
be 56.5% for
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
16 is within the range of values obtained for
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.
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 UnfoldingValues 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 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,
, ranges nearly in
parallel with the U50% values from 4.5 kcal/mol for
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.
We next examined the similarly stable
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
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.
|
For comparison with urea-induced unfolding of 16 in Fig.
7A, spectrin dimer was isolated from human red cells and its
urea-induced denaturation followed by [
]222 and by
tryptophan fluorescence in 0.1 M NaCl + 10 mM
Tris-HCl, pH 7.5, + 0.1 mM EDTA + 0.1 mM
-mercaptoethanol. Nearly identical with the average 66.7%
-helical content of
16, the
-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 [
]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
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.
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
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.
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
-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).
Mutation of the nearly invariant Trp-22 to tyrosine or
to phenylalanine, which are most frequently substituted for tryptophan in nature (37), reduces the
of
16
of chicken brain
-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
-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).
It seemed
possible that valine would be an adequate substitute for Trp-95 in
16, since valine occupies position 95 of
17 of chicken brain
-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
of W95V
than
16. In the crystal structure of the 14th repeating unit of
Drosophila
-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
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
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.
We compared urea-induced
unfolding of W22Y,S74L and 16, which exhibited similar
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
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.
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 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
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
16 and of human red cell spectrin dimer, 2.7 versus
3.4 M, respectively, indicate that the conformation of
cloned
16 could reasonably represent its conformation in a spectrin
dimer. (
-Spectrin of chicken brain is identical to
-spectrin of
avian red cells, since avian
-spectrin occurs as a single isoform
(39). Thus, it is reasonable to compare
16 of chicken brain
-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
of
unfolding of
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
of 6.4 kcal/mol but about the same 72%
-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
-spectrin have come to our attention (43, 44). Further investigation
is required to ascertain the structural significance of these
differences.
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 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).
In summary, the importance of Trp-22 for the thermodynamic stability of
16 and the similarity of the urea concentrations inducing
denaturation of 50% of
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.
We thank Dr. Matti Saraste for the generous
gift of chicken brain -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.