Structure and Function of Membrane Proteins. Proceedings of the International Symposium on Structure and Function of Membrane Proteins Held in Selva Di Fasano (Italy), May 23–26, 1983

362 Pages·1983·26.725 MB·English

by  Italy) International Symposium on Structure and Function of Membrane Proteins (1983 : Selva di Fasano, E. Quagliariello, F. Palmieri, E. Quagliariello

Developments in Bioenergetics and Biomembranes Volume 6
Other volumes in this series:
Volume 1 Bioenergetics of Membranes
Lester Packer, George C. Papageorgiou and Achim Trebst
editors, 1977
Volume 2 The Proton and Calcium Pumps
G. F. Azzone, M. Avron, J.C. Metcalfe, E. Quagliariello
and N. Siliprandi editors, 1978
Volume 3 Function and Molecular Aspects of
Biomembrane Transport
E. Quagliariello, F. Palmieri, S. Papa
and M. Klingenberg editors, 1979
Volume 4 Hydrogen Ion Transport in Epithelia
I. Schulz, G. Sachs, J.G. Forte and K.J. Ullrich
editors, 1980
Volume 5 Vectorial Reactions in Electron and Ion
Transport in Mitochondria and Bacteria
F. Palmieri, E. Quagliariello, N. Siliprandi
and E.C. Slater editors, 1981
STRUCTURE AND FUNCTION
OF MEMBRANE PROTEINS
Proceedings of the International Symposium on Structure and
Function of Membrane Proteins held in Selva di Fasano (Italy),
May 23-26, 1983.
Editors
E. Quagliartene
P. Palmieri
1983
ELSEVIER SCIENCE PUBLISHERS
AMSTERDAM · NEW YORK · OXFORD
© 1983 Elsevier Science Publishers B.V.
All rights reserved. No part of this publication may be
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Elsevier Science Publishing Company Inc.
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ISBN: for this volume: 0-444-80540-0
ISBN: for the series: 0-444-80015-8
Library of Congress Cataloging in Publication Data
International Symposium on Structure and Function of
Membrane Proteins (1983, : Selva di Fasano, Italy)
Structure and function of membrane proteins.
(Developments in bioenergetics and biomembranes ;
v. 6)
Bibliography: p.
Includes index.
1. Membrane proteins—Congresses. I. Quagliariello,
Ernesto. II. Palmieri, F. (Ferdinando) III. Title.
IV. Series. CDNLM: 1. Membrane proteins—Congresses.
2. Structure-activity relationship—Congresses. Wl
DE99TVK v.6 / QU 55 l6T^s 19833
QP552.MMtI57 1983 57^.87!5 83-16565
ISBN 0-UMi-805UO-0 (U.S.)
Printed in The Netherlands
V
PREPACE
The purpose of this International Sympsoium on "Structure and Function
of Membrane Proteins" at Selva di Fasano was to review the present
status of research into the structure-function relatioship of membrane
proteins. Structure was what the organizers had primarily in mind,
although they were aware that knowledge in this field is still embryonic.
However, membrane proteins are no longer those oily creatures floating
around in the lipid bilayer or in ill-defined detergent micelles. Mem­
brane proteins can be put into well-ordered arrays of either two-or
three-dimensional order, reflecting the well-defined monodisperse
structure of these entities. Wereas, prokaryotic membrane proteins,
particularly of the exotic varieties, are often highly expressed, making
solubilization and purification less important, more difficulties have
been encountered in the isolation of intact membrane proteins from
eukaryotic membranes. Only in the last ten years have these problems
been solved, in principle, through our understanding of the appropriate
handling of detergents, in particular of the non-ionic variety of these.
As a result, numerous sol ubi li zed membrane proteins are available in both
monodisperse and native forms.
It should now be possible to obtain more information on the structure
of these membrane proteins. Structural research comprises, first of all,
the elucidation of the amino acid sequence, the folding and the overall
shape, leading up to ultimate, atomic, resolution which requires crystals
suitable for X-ray crystallography. Progress in elucidating the primary
structure of membrane proteins has come from improved handling of the
hydrophobic peptides, and also from the use of cloned c-DNA sequences.
Two-dimensional arrays constituted the first step in ordering membrane
proteins and their successful application is now relatively widespread.
Three-dimensional crystals for X-ray crystallography are available in
only two instances, both of which have been discussed at this meeting.
From primary sequences, hypothetical folding predictions are made, with
the focus on the ubiquitous transmembrane α-helical segment. These
predominantly theoretical models can be useful for explaining sidedness
and assigning active sites, but will ultimately be replace by physical
data. The other approach to structure determination is based on
VI
monodisperse, soluble proteins or protein micelles using scattering
methods for X-ray, light and neutron beams. Structural information can
also be obtained by using fluorescence probes and spin labels as long as
specific localization and assignments at the protein can be made.
Structure and function are reciprocally linked. We wish to determine
the structure in order to understand the function, and the mechanism of
action will be understood only by our knowledge of the atomic structure.
The gathering of data on the function of membrane proteins prior to
knowledge of their structure is valuable for characterizing and defining
the proteins. Once the structure is known, another stage of research
will penetrate to the functional assignments of the structure.
We have witnessed in this symposium the various stages of this very
lively and effective research on biomembrane proteins. New enthusiasm
and scope for research has been created through obtaining the first
crystals of membrane proteins and it is to be hoped that some years from
now another meeting, possibly in the same room, might witness again the
great progress which we expect in this exciting field.
0 1983 Elsevier Science Publishers B.V.
Structure and Function of Membrane Proteins,
E. (luagliariello and F. Palmieri editors. 3
GRAMICIDIN A TRANSMEMBRANE CHANNEL: KINETICS OF PACKAGING IN
LIPID MEMBRANES.
MA SOT TI^, 2 ,
LANFFWNCO PAOLO CAVATORTA ALBERTO SPISNI~,E MANUELA
, 4
CASALIl, GIORGIO SARTORl, IVONNE PASQUALI-RONCHETT13 ARTHUR SZABO.
'Institute of Biological Chemistry, University of Parma, Viale
Gramsci, 14, 43100 Parma, (Italy); 21nstitute of Physics-GNCB,
University of Parma, Via D'Azeglio, 85, 43100 Parma, (Italy);
31nstitute of General Pathology, University of Modena, Via Campi,
165, 41100 Modena, (Italy); 'Division of Biological Sciences,
National Research Council, 100 Sussex Dr., Ottawa, Ontario K1A OR6,
(Canada).
INTRODUCTION
Biological membranes are recognized to act as anchoring point
for bound enzymes, to provide a medium where proteins, substrates
and products of enzyme reactions of limited solubility in water
become soluble, and where multienzyme complexes are organized. Fur-
thermore they define cellular compartments where substrates, prod-
ucts and effectors of metabolic reactions are separated and control
their selective transport between compartments. In particular, se-
lective ion movement across membraneis essential for the processes
of energy transduction and cell excitability.
Several attempts have been made to characterize native channels
in membranes in terms of structure and mechanism (1,2), but because
of their complexity, attention has also been focused on model sys-
tems capable to function as selective channels for ions. Gramicidh
A'(GA'), a hydrophobic polypentadecapeptide, has been shown to be
0
able to form helical structures with an internal pore of about 4 A
+
in diameter, that can accomodate monovalent cations such as Na or
.
K+ (3-10)
It has been demonstrated (11) that GA' can be incorporated into
lysolecithin micelles and that it assumes a left-handed helical
configuration able to transport monovalent cations selectively (12).
Moreover we have shown that the formation of the channel is associ-
ated with a reorganization of the lipid phase in a bilayer struc-
ture, wherein channel aggregates are embedded (13,14).
Having recognized the importance of a supramolecular organization
for the channel activity (15) our attention has been focused on the
4
studies concerning the kinetics of the incorporation and organiz­
ation of GA1 in the lipid phase.
MATERIALS AND METHODS
Lysolecithin was obtained from Sigma Chemical Company, St. Louis,
Mo., and checked for purity by nuclear resonance and thin layer
chromatography, Gramicidin was purchased from ICN Pharmaceuticals,
Cleveland, Ohio, as a mixture of 80% Gramicidin A, 6% Gramicidin
B, and 14% Gramicidin C, and was used without further purification.
The mixture is referred to as Gramicidin A1 (GA1).
Phospholipid was suspended in water and heated at 70°C up to
22 hours, as previously reported (15), either in the presence or in
the absence of GA1 . Two samples with different GA'/üpid ratios
were prepared: the first with 6 mg GA', the second with 3 mg GA'
per 25 mg of lysolecithin. Aliquots were withdrawn at times :
0, 10, 30, 60, 120, 180, 270 minutes and 21 hours.
Static fluorescence measurements were carried out as previously
described (15) . Lifetime measurements were performed using a
Spectra Physics apparatus with a pulsed laser source (1 psec).
Absorption measurements were performed using a Perkin Elmer
576 Spectrophotometer, equipped with a termostatically controlled
cuvette holder at temperature of 30±0.5°C. A molar extinction
coefficient of 22500 mol cm at 281 nm in CH^OH was used to
calculate the GA" concentration.
Circular Dichroism measurements were performed on a JASCO J-500
Spectropolarimeter equipped with a microprocessor unit for spectra
accumulation. The samples were diluted with water and run using
cuvettes with 0.2 mm pathlength. The ellipticities were calculated
using a mean molecular weight per residue of 124.5.
For electron-microscopic studies, the sample were left to equil­
ibrate at room temperature before being processed. The specimens
were examined in a Siemens Elmiskop 1A and Philips 410 electron
microscopes. The magnifications were calibrated by optical dif­
fraction of catalase crystals. For negative staining see caption
to Fig.4.
RESULTS AND DISCUSSION
In order to verify if the initial amount of GA1 used in the
5
experiments can influence the velocity of incorporation of the
polypeptide into the lipid system, the two concentrations re­
ported in the Materials and Methods section were chosen. The
percent of GA' incorporated, taking as 100% the actual initial
amount of the polypeptide, shows that the rate of incorporation
is somewhat proportional to the initial amount of GA1.
The fluorescence emission maximum, as shown in Fiq. 1, is shifted
from 342±1 nm at the time t=0 to 328+1 nm at the time t=22 hours
of incubation. Such a blue shift tipically indicates a change
in the polarity of the environment of tryOtophane residues, from
a polar to a non polar one.
— — — time zero
1 y^S. -'*-'-
\\ after 22 h
^ / ·;/
■\\
/ ·' '
b / ; ' v \\
< / ; ' \ *N
V-* / ·' ' \ "Λ
LU / ·* ; \ *A
Ü / ; '
zLU 1 : ; 1' \ \ *-*x·χ
ωÜ 0,5 / : / \ Λ
/ : ; \ #,·Ν
ce / .* ' \ *A
o / ; ; \ *"Λ
D / ; ' \ *
-J / : ' \ Λ
u. / ; f \ *Λ
;
1 · '
/ ·: ; X *·.\
/·"" '
// / ^ "***.
£·>
_j i ■*·■■ i 1
300 350 400 450
X(nm)
Fig. 1. Fluorescence spectra of GA1 incorporated into the lipid system at
different times of incubation.
The time course of absorbance, corrected for light scattering,
6
during incorporation, Fig. 2, clearly shows that the GA' incor­
porates into the lipid system. In fact at time t=0', the amount
of GA1 incorporated is just 6% of the total amount added and
reaches the 80% at t=22 hours.
At the same time the quantum yield decreases from a value of
0.7 for both the samples at time t=0', to 0.06 at t=22 hours.
However, the high values of the quantum yield at short times
might be overestimated, being the absorbance not entirely cor­
rected for light scattering and "obscured" absorbance (16).
O· 6 mg/ml
DB 3 mg/ml
60
-j
50 1.0
J
40 0.8
■ /
< Q.Y.
T /
oo
cc 30 0.6
O —a
</>
m 1 ö ^ ^~
J
20 -\ 0.4
J
10 0.2
σ · 1
n
200 400 600 800 1000 1200
TIME (min)
Fig. 2. Absorbance (open symbols)and quantum yield (Q.Y.)(full symbol)
dependence on time of incubation.
The CD spectra, shown in Fig. 3, demonstrate that also at the
time t=0', some of the polypeptide has already adopted an helical
structure. At subsequent times the CD pattern is similar to that
D
1.0
0.5
t-
0c
la a
4
9
-H
0.5
I
B 15
1.0
C 30
D 60
, ,
, I I ? , ,
190 210 230 2 50 2 70 290
h
tnm~
Fig. 3. Circular Dichroism spectra of G.A' incorporated into the lipid
systems at different times of incubation.
reported previously (111, until after 2 hours is consistent with
that of a left-handed helix, and it is stable with time.
The lifetime measurements (see Table 1) seem to indicate that
at the time t=O' two tvpes of tryptophane are detected, of which
one predominates. The lifetime does not change with wavelength
indicating that both types of tryptophane experience the same
environment. Up to 90' the lifetimes change very little. At
longer incubation times, shorter lifetimes are measured at 335
nm, while at longer wavelenqth again no change is observed.
The ultrastructural studies of the samnles durina incorworation