(Received for publication, April 14, 1995; and in revised form, August 7, 1995)
From the
The C-terminal fragment, residues 385-411, from human
fibrinogen -chain, i.e. KIIPFNRLTIGEGQQHHLGGAKQAGDV,
shows multiple turn conformations in aqueous solution (Mayo, K. H.,
Burke, C., Lindon, J. N., and Kloczewiak, M.(1990) Biochemistry 29, 3277-3286). The present study investigates the effect of
pH and trifluoroethanol on the conformation and backbone dynamics of
this 27-residue peptide. Both circular dichroism (CD) and
H-NMR data indicate the normally observed increased helical
conformations as a function of increasing trifluoroethanol.
H-NMR structural studies done in the presence of 40%
trifluoroethanol, pH 5.3, yield a network of nuclear Overhauser effects
consistent with significant populations of helix-like conformation.
Distance geometry calculations based on nuclear Overhauser
effect-derived distance constraints yield a family of structures with
relatively well defined N- and C-terminal conformations and an ill
defined mid-peptide region from Gly
to Gly
.
Similar conformational populations are observed at pH 2.5. CD studies,
however, indicate an increase in average
-helix content on
decreasing the pH from 6 to 2. This apparent conflict between CD and
NMR results may be explained by a transition from multiple
-turn
character at pH 5.3 to increased
-helix structure at pH 2.5.
C
NMR relaxation data analyzed with the
Lipari-Szabo model-free approach provide order parameters that
demonstrate little if any influence of pH on backbone motional
restrictions within the more flexible mid-peptide domain. At low pH,
however, motions become less restricted within N-terminal residues
Lys
-Phe
and more restricted within
C-terminal residues Ala
-Val
.
Protein folding is primarily dictated by noncovalent, relatively
weak intramolecular forces, i.e. hydrogen bonding,
electrostatic interactions, and hydrophobic effects (Jaenicke, 1991).
The effect of pH and alcohols on protein structure and folding has been
discussed widely in the literature. Depending on the protein, acids can
generate either fully or partially denatured states (Kuwajima, 1992).
For -lactamase, apomyoglobin, and ferricytochrome c (Goto et al., 1990), lowering the pH to about 2 by adding HCl yields
unfolded proteins whose conformations can be partially stabilized into
more compact states containing substantial secondary structure by the
addition of more HCl. More complex pH-induced folding transitions have
been observed at pH 2.7 with barnase (Sanz et al., 1994).
Alcohols have been known for some time both to denature/destabilize
globular protein tertiary (Conio et al., 1970; Parodi et
al., 1973) and quarternary (Yang et al., 1993) structure
and to effect conformational stabilization of various peptides in
aqueous solution (Conio et al., 1970; Parodi et al.,
1973). While some of these pH- and alcohol-induced states may be true
protein folding intermediates, the presence of alternatively folded
structures cannot be excluded. Nevertheless, their study can shed light
on general principles of protein folding and dynamics.
Recently,
more and more short linear peptides are being used as models for
protein folding and local structure formation. Although this approach
has been exemplified with synthetic peptides derived from the
-helix protein myoglobin (Waltho et al., 1993; Shin et al., 1993a, 1993b) and the mostly
-sheet protein
platelet factor-4 (Ilyina et al., 1994), studies on the
ribonuclease S peptide (20 residues) (Brown and Klee, 1971) and
pentapeptides like YPGDV (Dyson et al., 1988a, 1988b) first
fueled the fire of interest in others. NMR and CD, in particular, have
been used to show that short linear peptides can have considerable
conformational populations in aqueous solution in the presence and
absence of various stabilizing agents.
Trifluoroethanol is perhaps
the most commonly used agent for stabilizing -helix conformation
in peptides (Moroder et al., 1975; Lu et al., 1984;
Leist and Thomas, 1984; Dyson, et al., 1988a, 1988b; Pena et al., 1989; Lehrman et al., 1990; Segawa, and
Sugihara, 1984). Recently trifluoroethanol has been more thoroughly
studied in this function (Sönnichsen, et
al., 1992; Jasanoff and Fersht, 1994).
Sönnichsen et al.(1992) concluded that
trifluoroethanol is not a helix-inducing solvent, i.e. it does
not create new structures, but rather that it is a helix-enhancing
cosolvent that stabilizes helices in regions with existing
-helical propensity. The dominant effect of trifluoroethanol is
caused by its significantly weaker basicity with respect to that of
water (Llinas and Klein, 1975), which decreases amide proton hydrogen
bonding to the solvent and strengthens intramolecular hydrogen bonds,
thereby stabilizing secondary structure (Nelson and Kallenbach, 1986).
For some time, this laboratory has been interested in a peptide
derived from the C-terminal region of the fibrinogen -chain,
residues 385-411 (Mayo et al.(1990) and references
therein). During NMR and CD conformational studies of this 27-residue
peptide (called
27), (
)it was noticed that in the
presence or absence of trifluoroethanol, the average helix content
determined by CD was increased on lowering the pH from 6 to 2. On the
other hand, NOE magnitudes were decreased, suggesting the presence of
either less structure or increased internal mobility at lower pH. The
complications involved with interpreting NOEs from highly flexible,
linear peptides arise from the fact that NOEs are sensitive to both the
internuclear distance and internal motions. The present study was
initiated to correlate pH-induced CD and NOE effects in
27 with
motional characteristics derived from
C
H
relaxation experiments.
For sequential assignments, two-dimensional NMR-correlated spectroscopy (Aue et al., 1976; Wider et al., 1984), double quantum-filtered two-dimensional NMR-correlated spectroscopy (Piantini et al., 1982; Shaka and Freeman, 1983), and NOESY (Jeener et al., 1979; Wider et al., 1984) experiments were performed. Two-dimensional homonuclear magnetization transfer spectra, used to identify many spin systems completely, were obtained by spin locking with an MLEV-17 sequence (Bax and Davis, 1985) with a mixing time of 64 ms. All spectra were acquired in the phase-sensitive mode (States et al., 1982). The water resonance was suppressed by direct irradiation (1 s) at the water frequency during the relaxation delay between scans as well as during the mixing time in NOESY experiments.
The majority of the
two-dimensional NMR spectra were collected as 512 or 1024 t experiments, each with 1024 or 2,048 complex
data points over a spectral width of 5 kHz in both dimensions with the
carrier placed on the water resonance. 64 or 96 scans were generally
time-averaged per t
experiment. The data were
processed directly on the Bruker AMX-600 X-32 or offline on a Bruker
Aspect-1 work station using the UXNMR program. Data sets were
multiplied in both dimensions by 0-60°-shifted sine-bell or
lorentzian to gaussian transformation function and generally
zero-filled to 1,024 in the t
dimension prior to
Fourier transformation.
To obtain a quantitative description of
peptide backbone dynamics, H-detected
C
heteronuclear chemical shift correlation spectra (van Mierlo et
al.(1993) and references therein) were accumulated to derive (
H)-
C NOE and
C T
relaxation data on the unenriched peptide. In each case,
cross-peak intensities depend on the relaxation parameter of interest.
All spectra were acquired in the phase-sensitive mode by using
time-proportional phase incrementation for quadrature detection in the
dimension; 2048 data points were recorded in each
quadrature channel during t
, and 200 real points
were recorded in t
. Spectra were acquired with a
spectral width of 5000 Hz in
and 6000 Hz in
. The
H carrier was placed on the HDO
resonance, and the
C carrier was set at 45.4 ppm. For T
measurements, 128 scans were acquired per t
increment; for the NOE measurement, 256 scans
were acquired per increment. For measurements of T
and NOE, a relaxation delay of 5.0 s was used between scans to
ensure sufficient recovery of
H magnetization. For T
relaxation measurements, nine separate spectra
were recorded for T = 0.01, 0.04, 0.08, 0.15, 0.2, 0.3,
0.6, 0.8, and 1.2 s. Relaxation rate constants and NOE enhancements
were calculated from peak heights of the heteronuclear resonances as
described by Palmer et al. (1991). Data analysis was performed
on Bruker Aspect-1 or Silicon Graphics work stations using UXNMR,
Aurelia (Bruker, Inc.), or FELIX (Biosym, Inc.) programs.
C NMR relaxation data were analyzed by using the
model-free formalism of Lipari and Szabo (1982a, 1982b) in which
motions are described in terms of two correlation times (an overall
tumbling time and an internal motion correlation time) and an order
parameter (S
), which can be related to bond
angular restrictions (Lipari and Szabo, 1982b). Since S
is least sensitive to
, an average
value of 5
10
s was used initially in
the optimization routine and later varied up to 10
10
s. Then a resulting average
value was fixed, and
and S
were allowed to vary. In either case, S
varied by no more than 5%. Smaller values of the order parameter
are taken to indicate relatively decreased motional restrictions.
Figure 1:
CD spectra of 27. CD
spectra of
27 obtained at pH 6 and 5 °C are shown at different
trifluoroethanol concentrations as indicated in the figure.
Trifluoroethanol concentrations are given as percentage (v/v) of
composition. The peptide concentration was kept approximately constant
at 40 µM. Based on CD data according to , the
calculated
-helix content in
27 is plotted versus trifluoroethanol concentration in the inset. In the inset, data are shown for pH 6, 5 °C, and 25 °C. Lines drawn through points are for visual aid
only.
where is the observed mean residue ellipticity at 220-nm
wavelength.
is the maximum mean residue ellipticity
of a helix of infinite length (Chang et al., 1978); f
is the fraction of helix in the molecule; i is the number of helical segments; N is the total number
of residues, and k is a wavelength-dependent constant (2.6 at
220 nm). The number of helical segments, i, was set to 2 in
order to be consistent with modeled structures generated from
NOE-derived distance constraints discussed later. The expected value of
the mean residue ellipticity for 100% helicity of peptides of chain
length 27 residues was determined to be -32,720 deg
cm
/dmol. Using this method, calculated helicities as a
function of trifluoroethanol concentration are shown in the inset to Fig. 1for data accumulated at 5 and at 25 °C. As
expected, helicity increased with increasing trifluoroethanol
concentration and with decreasing temperature.
CD spectra are
plotted as a function of solution pH (constant 40% trifluoroethanol and
5 °C) in Fig. 2. In the insert to Fig. 2,
[], [
], and
the wavelength minimum in the 204-206-nm range, are plotted versus the solution pH. These CD data (inset) are
consistent with increases in the average helix content of peptide
27 as the pH is lowered from 6 to 2. Even though data shown in
this figure have been accumulated in 40% trifluoroethanol, the same
general trend is observed at lower trifluoroethanol concentrations. The
isodichroic point at 200 nm supports the idea of an equilibrium between
two main conformational populations, one having more helix character
than the other.
Figure 2:
pH
effect on CD spectra. CD spectra for 27 are plotted versus the solution pH from pH 2 to 6. In the inset, molar
ellipticities are plotted for two wavelengths, 195 and 220 nm, as a
function of the solution pH. At the top of the inset,
wavelength in the 204-206-nm range is plotted versus the
solution pH. trifluoroethanol concentration was constant at 40% (v/v).
The temperature was 5 °C. Data points in the inset indicate the simple average of three pH titration series. Standard
deviations are ±0.5
10
deg cm
dmol
at 220 nm; ±2
10
deg cm
dmol
at 195 nm; and
± 0.2 nm for the wavelength versus pH plot. Lines connecting datapoints are for visual aid
only.
Figure 3:
pH 5.3
NOESY contour plots of peptide 27. The
H-NH/aromatic and
NH-NH/aromatic resonance regions from a NOESY contour plot are shown.
Data were collected in 60%
H
O/40% perdeuterated
trifluoroethanol (0.6-ml total sample volume) with 10 mM peptide
27 at pH 5.3 and 5 °C. 512 hypercomplex free
induction decays containing 1024 words were collected and processed as
discussed under ``Materials and Methods.'' The mixing time
was 0.1 s. The data were zero-filled to 1024 in t
.
The raw data were then multiplied by a 40°-shifted sine-squared
function in t
and t
prior to
Fourier transformation. Some sequential resonance assignments are
traced out, and some longer range NOEs are indicated. Labeling of
resonances is as discussed in the text.
Figure 4:
pH 2.5 NOESY contour plots of peptide
27. The
H-NH/aromatic and NH-NH/aromatic resonance regions
from a NOESY contour plot are shown for data accumulated at pH 2.5.
Data collection and processing was as discussed in the legend to Fig. 3and in the text. Resonances are labeled as discussed in
the text.
Figure 5:
Summary of NOE data for peptide 27.
The peptide sequence of
27 is shown with a summary of identifiable
NOEs given above for data accumulated at pH 5.3 and below for data accumulated at pH 2.5. NOEs are tabulated in the format
discussed by Wüthrich(1986). A questionmark indicates ambiguity in identifying a possible
NOE.
Distance constraints were
derived from NOEs listed in Table 2and were used in distance
geometry calculations for conformational populations of 27 at pH
5.3, 40% trifluoroethanol, 5 °C. The time dependence of NOEs was
used to check for possible spin diffusion. Below about a 0.5-s mixing
time in the NOESY experiment, spin diffusion could not be detected.
NOEs were ranked relatively as strong (2.2-3 Å), medium
(2.8-3.5 Å), and weak (3.3-5 Å). An additional
0.5 Å degree of freedom was allowed for each non-backbone atom
(or pseudoatom) involved in any given distance constraint. Distance
geometry calculations were first done by using XPLOR, followed by
energy minimization and restrained annealing dynamics simulations.
Electrostatic potentials for charged groups were varied from full
charge to about 50% of full charge. 30 structures were generated. 10 of
these showed minimal distance violations (from input NOE constraints)
of less than 0.5 Å. Overall backbone RMSD values were less than
0.8 Å
for residues
Phe
-Glu
and for residues
Glu
-Asp
. Fig. 6displays two
sets of the same 10 structures generated this way. The leftportion of the figure shows overlays for the N-terminal
segment residues 389-396, and the rightportion of the figure shows overlays for the C-terminal segment residues
404-410. Both N- and C-terminal segments form helix-like
conformations.
-Helix character is greatest for sequences
Arg
-Gly
and
Ala
-Gly
. Ramachandran plots (data not
shown) indicate that the greatest
,
angular displacements
are found for Gly
-Gly
. In this
respect, it appears that the terminal segments move more or less as
units connected via a mid-segment ``hinge'' region.
Figure 6:
Computer-modeled structures of 27.
Based primarily on NOE-derived distance constraints, distance geometry,
restrained minimization, and dynamics, simulated annealing calculations
were performed using the XPLOR program on an SGI 480 computer. The
superposition of backbone atoms of 10 structures are shown as discussed
in the text. On the leftside of the figure,
the best overlay for N-terminal residues is shown, while on the rightside, the best overlay for C-terminal residues
is shown. In this figure, residues are labeled from 1 to 27
instead of from 385 to 411.
Figure 7:
Chemical shift versus pH for
His and His
C2. Chemical shifts for
His
and His
C2 proton resonances are
plotted versus the solution pH. The insert expands the
chemical shift ordinateaxis to better display the
lower pH inflection in the histidine titrations curves. Data for
His
alone are shown connected by the solidline. Solid and opensquares indicate calculated titration curves for Glu
with a
pK
of 4.1 and for His
with
a pK
of 6.6. These two theoretical curves
sum up to yield the solidline drawn through the
His
data points.
Significant pH-dependent chemical shift changes
(greater than 0.1 ppm) for side chains of Lys,
Ile
, Arg
, and Thr
(data not
shown) also argue for direct (although probably transient) interactions
with Glu
. In a helix-like conformation (Fig. 6),
Thr
is located at the i, i + 3
position with respect to Glu
. Proximity to N-terminal
residues Lys
and Ile
is considered
plausible based on results from calculated structures. In particular,
the side-chain of Lys
can fold in toward the side-chain
of Glu
to mediate a ``loose'' electrostatic
interaction. Within the Lys
-His
segment, 50% of these backbone NHs are shifted more than 0.1 ppm
on varying the pH between 2.5 and 5.3. In particular, two of the more
shifted NHs belong to Ile
and Phe
,
supporting the idea of a possible long range structurally stabilizing
effect of Glu
. Additionally, Ile
NH is one
of the most long lived NHs at lower pH (Mayo et al., 1990).
This electrostatic interaction in combination with hydrophobic
side-chain clustering (Dyson et al., 1992), could explain this
NH solvent protection.
Protonation/deprotonation of Asp and Val
(C-terminal carboxylate) probably plays no
role in the electrostatic effects of the N-terminal and mid-peptide
segments. The only side chain within C-terminal residues
Leu
-Val
that shows significant
chemical shifts on varying pH belongs to Ala
. Other
residues have their backbone resonances more highly shifted than their
side-chain resonances, suggestive of indirect, conformationally induced
chemical shift changes. In particular, the noncharged amino acid
residue NHs of Gln
, Ala
, and Gly
are shifted by between 0.15 and 0.25 ppm, and Gly
Hs are more degenerate at lower pH. Interestingly,
side-chain proton resonances of Lys
demonstrate a
carboxylate pK
inflection. This suggests an
electrostatic interaction between Lys
and most probably
Asp
or Val
.
Figure 8:
(H)-
C Hetcor NOE
Data. Two
H-
C heteronuclear shift-correlated
NOE data sets (van Mierlo et al., 1993) are shown for
27
at pH 5.3 (A) and pH 2.5 (B). Resonances have been
assigned as discussed in the text.
Figure 9:
27
C T
, NOE, and order parameters.
C T
, (
H)-
C NOE data, and
backbone motional order parameters, S
, for
27
at pH 2.5 and at pH 5.3 are shown. As discussed under
``Materials and Methods'' and in the text, order
parameters, S
, have been derived from these
relaxation data by using the model-free approach of Lipari and Szabo
(1982a, 1982b).
Within the N-terminal
segment Lys-Phe
, both T
and NOE values are smaller for
27 at pH
5.3, indicating decreased backbone mobility of that sequence at higher
pH. This is reflected in order parameters, S
,
derived from these relaxation data (Fig. 9). For residues
Ala
-Val
, S
values are
larger at pH 2.5, indicating increased motional restriction of the
C-terminal segment at lower pH. For residues
Asn
-His
, S
values vary
less with pH change. At either pH, the mid-peptide region residues
Thr
-His
generally show the smallest
order parameters, indicating the presence of a relatively flexible
mid-peptide segment, consistent with NOE-based distance geometry
calculations, which indicate an ill-defined mid-peptide segment from
about Gly
to His
. The overall correlation
time,
, was 1.9 ns at either pH value.
Short, linear peptides, like 27, generally exist in
solution in an ensemble of highly fluctuating structures whose NMR
spectral parameters average. This is true for
27 in aqueous
solution at 30 °C in the absence of trifluoroethanol (Mayo et
al., 1990) where multiple turn or nascent helix (Dyson et
al., 1988a, 1988b) conformation was apparent within residues
385-402. Under those solution conditions, C-terminal residues
402-411 showed no NOE structural constraints greater than i, i + 1; however, conformational preference
within that segment was apparent based on chemical shift differences
with fibrinogen
-chain peptide 400-411 and a 5-Hz
J
coupling
constant for Ala
(Mayo et al., 1990). This
observation is supported with NMR studies on fibrinogen
-chain
peptide 392-411 done by Blumenstein et al.(1992), who
reported that at 5 °C (also in the absence of trifluoroethanol), a
significant
-turn population exists for the sequence
Gln
-Asp
.
These present 27 NOE
data accumulated in the presence of trifluoroethanol are consistent
with both reports (Mayo et al., 1990; Blumenstein et
al., 1992). More transient multiple turn or helix-like
conformations noted at 30 or 5 °C in the absence of
trifluoroethanol are stabilized by the presence of trifluoroethanol,
which acts as a structure-enhancing cosolvent
(Sönnichsen, et al., 1992; Jasanoff and
Fersht, 1994), rather than as a conformation-inducing, i.e. new structure-inducing, agent. Trifluoroethanol stabilizes helix
conformation in peptide sequences that have some helix propensity. The
Chou-Fasman(1978) predictive secondary structure algorithm yields good
probabilities for helix formation from residues
Leu
-Leu
as well as from residues
Ala
-Val
(Mayo et al., 1990).
At pH 5.3, NOE-based distance geometry-generated structures of
27
indicate that helix-like or multiple turn conformations are present
within the N- and C-terminal segments, residues 391-397 and
404-408, respectively. N-terminal residues 385-387 have an
extended conformation, and Pro
causes a kink in the
structure that leads into a turn centered at 390-391. The
trifluoroethanol-stabilized, N-terminal conformation, residues
385-397, is essentially the same as that observed for
27 in
aqueous solution at 30 °C (Mayo et al., 1990), once again
supporting the idea that trifluoroethanol does not induce new structure
formation but rather acts to enhance existing conformational
populations (Söennichsen et al., 1992;
Jasanoff and Fersht, 1994). Within the
Gly
-Gly
segment, few ``long
range'' NOEs are observed, which results in distance geometry
calculations of a conformationally ill defined mid-peptide region. The
paucity of NOEs could be the result of a more extended, solvent-exposed
conformation and/or of a more flexible domain. Since average motional
order parameters are reduced within this region relative to other
sequences, one can conclude that the mid-peptide segment is relatively
more flexible than any other segment. The lack of conformationally
constraining NOEs within this region, therefore, is mostly due to the
presence of an ensemble of highly fluctuating conformations. In this
respect, N- and C-terminal helix-like regions are connected by a
``hinge'' segment. In support of this, it should be noted
that glycine, which highly populates this mid-peptide segment
(Gly
, Gly
, Gly
,
Gly
), normally promotes increased
,
angular
freedom and flexibility, disrupts periodic structure, and frequently
occupies the helix C-cap position (Richardson and Richardson, 1988).
In terms of the effect of pH on specific sequences within 27,
NMR data indicate that N- and C-terminal domains behave differently.
Generally, the same NOEs are observed at either pH 2.5 or pH 5.3,
indicating the presence of similar conformational populations. NOE
magnitudes at pH 2.5, however, are reduced on average by about
10-20% relative to those observed at pH 5.3. Most NOE magnitudes
observed within the mid-peptide region are unaffected by pH changes.
Within the N-terminal segment, which becomes more flexible at lower pH,
however, NOE magnitudes are generally reduced, suggesting a more
``open'' or less structured
27 N-terminal conformation
at pH 2.5. This is consistent with results from protein folding studies
where decreasing the pH to 2-3 denatures or unfolds protein
structures. Consistent with distance geometry structural calculations,
Lys
and the N-terminal amine may interact
electrostatically with Glu
; and by neutralizing
Glu
by lowering pH these charge-charge interactions are
minimized or negated, causing the N-terminal segment to become less
conformationally and dynamically restricted, resulting in reduced NOE
magnitudes.
Unlike the N-terminal domain, the C-terminal segment,
residues Ala-Val
, becomes more motionally
restricted at lower pH. In apparent contradiction to this, NOE
magnitudes, particularly those of NH-NH, are reduced for these
C-terminal residues, while the change in CD molar ellipticity
translates into an approximately 15% increase in average helix content.
For short linear peptides that exist in a highly dynamic conformational
ensemble that displays some average ``structure,'' NOEs are
difficult to interpret since they are affected both by changes in
internuclear distances and by motional properties of the peptide.
Increased negative CD ellipticities at 224 nm could be the result of
increased
-turn character at the higher pH value, which would show
a more positive absorption at 224 nm and would reduce the apparent
negative ellipticity at 220 nm (Dyson et al., 1988a and
1988b). In this respect, these results suggest that the
27
conformational ensemble is shifted to a more helical character at lower
pH. Reduced NH-NH NOE magnitudes, for example, would be explained by
increased average NH-NH internuclear distances in a helical
conformation relative to a tight turn.
In conclusion, this study has
shown that for the more hydrophobic N-terminal segment that may be
partially stabilized by electrostatic interactions, lowering the pH
induces a more open, more dynamic conformational ensemble, while for
the C-terminal segment lowering the pH shifts this ensemble to a more
helical, less flexible conformational distribution. For 27, pH has
the effect of acting at the local, rather than global, conformational
level.
This paper is dedicated to the memory of Simon Pilkis, chair of the Biochemistry Department, who died suddenly August 2, 1995.