(Received for publication, July 10, 1995; and in revised form, September 20, 1995)
From the
The effect of membrane binding on the structure and stability of
several conformers of -lactalbumin was studied by infrared
spectroscopy, circular dichroism, and fluorescence spectroscopy. In
solution, under experimental conditions where all conformers interact
with negatively charged membranes, they show significant conformational
differences. However, binding to negatively charged membranes, which
causes considerable changes in the structure of these conformers, leads
to a remarkably similar protein conformation. The membrane-associated
conformations are characterized by 1) a high helical content, greater
than any of those found in solution, 2) a lack of stable tertiary
structure, and 3) the disappearance of their thermotropic transition.
These observations indicate that association with negatively charged
membranes induces a conformational change within
-lactalbumin to a
flexible, molten globule-like state.
There is a large class of proteins (and peptides) which, although soluble in water, during the course of their action interact with plasma or intracellular membranes(1) . The transition from the water-soluble to the membrane-associated state is believed to involve large structural changes(1, 2, 3, 4) . The nature of this conformational change is a central issue to understand the problem of protein folding in membrane environments. In this context, the important role of lipid-protein interactions in protein translocation across biological membranes and protein insertion into lipid vesicles has been recognized both in vivo and in vitro(5, 6, 7) . The use of phospholipid biosynthetic mutants in Escherichia coli has revealed that anionic lipids are essential for efficient membrane insertion and translocation of newly synthesized proteins through both the ``Sec''-dependent and -independent pathways(8) . Investigations on simple defined model membranes have shown that anionic lipids are required for membrane insertion and translocation of proteins(9, 10, 11) . Changes in protein structure, facilitated by interaction with negatively charged phospholipids, allows membrane insertion and subsequent translocation of proteins(8, 9, 10, 11, 12) . The experimental difficulties encountered in conformational studies of membrane-associated proteins have led to the present lack of knowledge on the structural transition involved in such association processes.
To further examine the effect of membrane binding on protein
structure we focus in this study on the interaction of native and
several folding intermediates of -lactalbumin (
LA) (
)with model membranes.
LA is able to interact with
lipid bilayers (13, 14) and provides a suitable model
to characterize the structural transition involved in membrane
association of water-soluble proteins, since its conformational
properties have been extensively studied(15, 16) .
Moreover, the possibility of trapping stable reduction intermediates of
this protein offers the opportunity to follow the conformational
changes associated with binding of different folding intermediates to
membranes(16, 17) . As experimental techniques we use
infrared (IR) spectroscopy, circular dichroism (CD), and fluorescence
spectroscopy. The complementary information provided by these
biophysical methods reveals that binding to negatively charged
membranes induces a different structural change in the
LA
conformers which leads to a very similar, membrane-bound flexible
conformation.
Figure 1:
Original (A) and deconvoluted (B) infrared spectra of LA and its derivatives in
solution(- - - -) and in the presence of PC:PG (1:1)
LUV at a lipid to protein molar ratio of 300:1 (-). Trace 1, holo-
LA; trace 2, apo-
LA; trace 3, holo-3SS
; trace 4,
apo-3SS
. The spectra were recorded in D
O
media (20 mM Na
PO
, 100 mM NaCl (pD 4.5)) containing 1 mM Ca
(holobuffer) or 1 mM EDTA (apobuffer) at 25 °C.
Protein concentration was 0.7 mM. Deconvolution was performed
using a Lorentzian with half-bandwidth of 18 cm
and
a band-narrowing factor of 2.
Another important difference, clearly evidenced in Fig. 1B, concerns the bands located at 1576 and 1590
cm which come from the antisymmetric
COO
stretching mode of the aspartic and glutamic
residues(24) . The intensity of these bands is drastically
reduced on membrane association, indicating that neutralization of most
of the protein acidic residues is required for the interaction to
occur. Unfortunately, the bands corresponding to the -COOH
protein groups, which normally appear between 1700 and 1750
cm
, cannot be detected since they overlap with the
strong absorption band of the phospholipid ester groups (1730
cm
; Fig. 1A).
To gain insight
into the structure of LA bound to lipid vesicles, we have compared
the thermal stability of the different conformers in solution and
associated with lipid vesicles, under identical experimental conditions (i.e. pH 4.5). The temperature-induced changes observed in the
amide I band of these conformers in solution are similar to those
reported previously for soluble and membrane
proteins(22, 23) ; namely, a loss of regular secondary
substructures and the emergence of band components at 1618
cm
and 1681 cm
, which have been
assigned to hydrogen-bonded extended structures formed upon aggregation
of thermally denatured proteins (data not shown). As a consequence of
these conformational changes, the amide I band undergoes an
irreversible cooperative broadening with increasing temperatures (Fig. 1). Therefore, protein thermal unfolding can be easily
followed by plotting the amide I bandwidth at half-height versus temperature. The ``melting curves'' obtained for the
apo- and holonative proteins indicate that both undergo thermotropic
transitions in solution (Fig. 2). In contrast, there is no
evidence of a cooperative transition for the same membrane-associated
conformers. Instead, the width of their amide I band follows a small,
progressive temperature-induced increase similar to that obtained for
their acid compact intermediate (Fig. 2). Identical results were
obtained for the 3SS
conformers (data not shown).
Figure 2:
Temperature dependence of the amide I
bandwidth at half-height (BWHH) for holo- (closed
symbols) and apo-LA (open symbols) at pD 4.5 in the
absence (circles) and presence of PC:PG (1:1) LUV (squares), and in solution at pD 2.5 (triangles).
Protein concentration was 0.7 mM.
To
further explore the molten globule-like thermal stability of the
membrane-bound conformers, we have compared their IR spectra with those
of their corresponding acid compact intermediates (Fig. 3). Note
that the terms molten globule and compact intermediate are used
interchangeably. The deconvoluted spectrum of the acid molten globule
of native, holo-LA exhibits two main components at 1650 and 1639
cm
and a broad feature at around 1680
cm
which are assigned as above (Fig. 3, trace 1). The modest downward shift observed for the main
components, as compared with their positions in solution and in the
membrane-bound state, reflects an increased accessibility of the
protein backbone to solvent exchange. The presence of a less intense
residual amide II band at 1545 cm
, partially due to
unexchanged N-H groups, in the spectrum of its acid molten
globule state corroborates this interpretation ( Fig. 1and Fig. 3). The width of the major components is also broader in
the acid compact intermediate, suggesting an increase in the
conformational motion of the different secondary structure elements.
Apart from these differences and in agreement with previous structural
studies(25, 26) , the main features of native
holo-
LA secondary structure are essentially preserved in its acid
molten globule state. A comparison of the IR spectra corresponding to
the acid molten globule states of the different conformers shows that
while those of the native holo- and apoproteins are very similar (Fig. 3, traces 1 and 2), the intensity at
around 1631 cm
is slightly and clearly stronger in
the spectra of holo- and apo-3SS
, respectively (Fig. 3, traces 3 and 4). This difference
could reflect a higher propensity to self-associate, at the relatively
high protein concentration used for IR spectroscopy, of their acid
compact intermediates. The fact that at 60 °C only the spectra of
the 3SS
conformers display band components at 1618 and
1684 cm
, indicating protein aggregation, supports an
increased exposure of hydrophobic regions in their acid states (data
not shown). This finding fits in with the existence of molten globule
states with different conformational flexibility, as recently proposed
by Redfield et al.(27) . Remarkably, none of the above
mentioned differences are seen between the membrane-bound conformers.
Figure 3:
Deconvoluted infrared spectra of the
membrane-bound (-) and acid compact intermediates(-
- - -) of LA and its derivatives. Trace
1, holo-
LA; trace 2, apo-
LA; trace 3,
holo-3SS
; and trace 4, apo-3SS
.
The pD of the lipid containing samples (lipid/protein ratio, 300:1) was
4.5 while that of the acidic molten globule states was 2.5. Other
details as in Fig. 1.
Figure 4:
Far-UV CD spectra of the different LA
conformers in solution (traces 1-4) and bound to PC:PG
(1:1) LUV at a lipid to protein molar ratio of 300:1 (traces
5-8). Native
LA (traces 1 and 2),
3SS
(traces 3 and 4). Spectra were
taken at 25 °C in 20 mM Na
PO
, 100
mM NaCl (pH 4.5), containing 1 mM Ca
(traces 1 and 3) or 1 mM EDTA (trace 2 and 4). Protein concentration was 24
µM. For clarity, the spectra of the membrane-associated
conformers have not been specifically
numbered.
The CD spectrum
in the near-UV region of native holo-LA in solution shows a
pronounced negative ellipticity at around 270 nm and a positive peak at
293 nm which have been assigned to Tyr and Trp residues, respectively (Fig. 5, trace 1)(29) . This spectrum is
characteristic of a native-like conformation with a fixed orientation
of the
LA's 12 aromatic residues, which are spread
throughout the protein molecule(26) . Inspection of the spectra
corresponding to the other conformers indicates that the environment of
the aromatic side chains becomes less rigid in the following order:
holo-
LA > holo-3SS
> apo-
LA
apo-3SS
(Fig. 5, traces 1-4).
Interestingly, the spectra of the four conformers associated with
negatively charged LUV are similar, lacking the fine structure
characteristic of the native structure in solution (Fig. 5, traces 5-8). The general shape of these spectra
resembles that of the ``collapse'' solution spectrum at
acidic pH(15) .
Figure 5:
Near-UV CD spectra of native LA (traces 1 and 2) and its 3SS
derivative (trace 3 and 4) in solution (traces
1-4) and bound to negatively charged vesicles (traces
5-8). Protein concentration was 17 µM. Other
details are as described in the legend to Fig. 4.
Figure 6:
Effect of negatively charged membranes on
the tryptophan fluorescence of LA and its derivatives.
Measurements were performed at 25 °C in 20 mM
Na
PO
, 100 mM NaCl, containing 1
mM Ca
(A) or 1 mM EDTA (B). Traces 1, 3, and 5, native
LA; traces 2, 4, and 6,
3SS
. Spectra were recorded at pH 4.5 in the absence (traces 1 and 2) and presence of PC:PG (1:1) LUV at a
lipid to protein molar ratio of 300:1 (traces 3 and 4), and in solution at pH 2.5 (traces 5 and 6). Protein concentration was 1.1
µM.
The results of this study demonstrate that the membrane-bound
conformation of different LA intermediates are remarkably similar,
in spite of showing different structural properties in solution. The
protein conformational changes induced on membrane binding may be
summarized as follows.
Taken together, these results indicate that
negatively charged membranes bind a reduced number of similar highly
helical and flexible protein conformations, which share structural
properties with the acid molten globule state and the GroEL-bound
protein(35) . The fact that the conformations of the
membrane-bound and acid compact intermediate states of LA and its
derivatives are not identical supports the view of the molten globule
state not as a single conformation but rather as a family of more or
less ``ordered'' conformers(27) .
The membrane-bound, highly dynamic competent state, distinct from the native or aggregated state, could facilitate protein insertion into and/or protein translocation across membranes, assembly of membrane protein complexes, or proper interaction of the protein with the transport machinery for its subsequent translocation. This would be consistent with the observation that protein translocation requires partial unfolding of its mature part(36) . Negatively charged membranes could play a complementary role to that exerted by the variety of proteins which are directly or indirectly involved in protein translocation(37) , as it has been proposed recently for the mitochondrial protein import(36) .