| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Biophysical Journal 74: 1-2 (1998)
© 1998 the Biophysical Society
Biophys J, January 1998, p. 1-2, Vol. 74, No. 1
Department Physical Biochemistry, Institute of Biochemistry, Darmstadt University of Technology, Darmstadt, Germany
Bacteriorhodopsin in the purple membrane of
halobacteria is a prominent prototype of an integral membrane protein,
especially of the family of seven membrane-spanning Tracking the individual steps of charge translocation through
bacteriorhodopsin obviously requires knowledge of its structure. Fortunately, by high-resolution electron crystallography on natural, two-dimensional purple membrane crystals (Kimura et al., 1997 How are these conformational changes in bacteriorhodopsin coupled to
the vectorial, electrogenic proton transfer from the cytoplasmic side
to the extracellular side? The kinetics, location, and
stoichiometry of proton transfer reactions, such as proton ejection to
and migration along the membrane surface as well as into the aqueous
water phase, have been successfully determined by the application of
laser absorption spectroscopy with pH probes either covalently bound to
bacteriorhodopsin or residing in the bulk (Heberle et al., 1994
ARTICLE
-helical
proteins. At present, this light-driven proton pump is one of the best
characterized active ion-translocating proteins. Soon, it will be the
first membrane protein whose vectorial transport mechanism is
understood at the molecular and even atomic level. To unravel the
mechanism of a catalytic protein, such as bacteriorhodopsin, the link
between the structure, its dynamics, and the function has to be
established. The concerted application of both powerful biophysical
methods and tailor-made bacteriorhodopsin samples shall make it
possible to reach this goal. The report by Nagel and co-workers (1997)
in this issue is a marvelous example of that; indeed, it is a successful way to approach the goal.
) as well
as by x-ray diffraction on three-dimensional (3D) crystals (Pebay-Peyroula et al., 1997) the 3D structure of bacteriorhodopsin has
been recently determined at a resolution of 3.0 and 2.5 Å, respectively, supporting the pioneering work of R. Henderson and co-workers. Soon, we can expect a bacteriorhodopsin structure at 2.0 Å. However, to pinpoint the elementary steps of proton translocation,
the bacteriorhodopsin structure of the ground state is not sufficient.
The structure of the photocycle intermediates, at least of the
functionally relevant intermediates is also required. Although
controversial at the beginning, the light-induced appearance of
tertiary structural changes in the protein, predominantly at helix F
and G, has now been established by various investigations using
electron crystallography and neutron and x-ray diffraction. The
photocycle and the proton pumping cycle of bacteriorhodopsin are
accompanied and most probably propelled by structural changes in both
the chromophore, the protonated Schiff base of retinal, and the protein
(Hauss et al., 1994). Recent structural studies on wild type and mutant
bacteriorhodopsin have revealed that the tertiary structural changes in
the protein moiety occur in the M intermediate and persist also in the
N intermediate. The onset of these tertiary structural changes is
correlated with the transition between two structurally distinct
substates of the spectroscopic intermediate M, designated
M1 and M2 (Sass et al., 1997; Nagel et al.,
1997). These large changes in the structure take place after the
transfer of the proton from the Schiff base to the amino acid Asp-85
and after the release of the proton to the extracellular surface. This
indicates that these structural changes follow a redistribution of
charges. (It should be noted that a different view has been published
in some Fourier transform infrared studies, i.e., the onset of the
largest structural changes, as solely defined by difference bands in
the amide I and II regions, in the transition from M to N.)
). In
contrast, charge translocation through the core of the protein is best
studied by electrical measurements. During the past 25 years, several
approaches to monitor light-induced charge translocation across the
purple membrane have been applied, which are marked by their extreme
sensitivity and often by their high time resolution. However, the
interpretation of the data of most studies dealing with the
lightinduced charge translocation across bacteriorhodopsin and of
all studies examined the voltage dependence was severely hampered by
the undefined and non-unidirectional orientation of the current
generator bacteriorhodopsin. In this respect, the study by Nagel et al.
(1997) is unique. For the first time, the voltage dependence of charge
translocation by the proton pump could be unequivocally determined,
i.e., under well defined voltage clamp conditions, by expressing
bacteriorhodopsin from Halobacterium salinarium in the
plasma membrane of oocytes from Xenopus laevis and by
measuring the current voltage behavior of this unidirectionally
oriented bacteriorhodopsin population. According to the interpretation
of the voltage dependence of the pump current and blue light quenching
effect, the externally applied electrical field changes the ratio of
the M1 and M2 states. This should also occur
during light-induced generation of an electrochemical gradient in the
halobacteria. The observation that negative potentials arrest the
protein in M1 is of functional relevance, yet it could also
be an experimental tool to study this substate, e.g., by diffraction
techniques or Fourier transform infrared spectroscopy. The voltage
dependence of the ratio between M1 and M2 is a
strong hint for the functional importance of these two M substates in controlling the vectoriality of the proton pump. During formation of
M1, a proton is transferred from the Schiff base to Asp-85 and at the same time, one proton is ejected to the extracellular surface of bacteriorhodopsin. In the M2 to N transition,
the Schiff base becomes reprotonated, but via the cytoplasmic side,
receiving its proton from Asp-96, whose pK is lowered. Thus, the
spectroscopic intermediate M is, in fact, composed of two structural
different substates that differ in the accessibility of the Schiff base for protons and its pK (intermediate M1: Schiff base
accessible to the extracellular proton pathway, pK shifted from
originally >13 to <4; M2: Schiff base accessible to the
cytoplasmic proton pathway, pK >10). The important finding by Nagel et
al. (1997) is that the electrical potential regulates the ratio between
M1 and M2 in this key transition, which might
be the sole or at least a "molecular switch" in the pumping
mechanism (another molecular switch could be the observed positional
change of the retinal polyene chain close to the Schiff base; Hauss et
al., 1994). The electrical measurements on membranes with perfectly
oriented bacteriorhodopsin molecules can bridge the gap between proton
translocation steps through this pump and the light-triggered tertiary
structural changes. This experimental system would be the perfect one
to unravel the elementary steps of proton translocation if
spectroscopic and structural investigations would be feasible. However,
because the density of bacteriorhodopsin expressed in the plasma
membrane is relatively low, not even the photocyle can be studied at
present. Presumably bacteriorhodopsin is in the monomeric state and
highly mobile as opposed to the trimeric state in the crystalline
purple membrane. This might be even advantageous for data
interpretation, as the bacteriorhodopsin monomer is the functional unit
for proton pumping.
| |
FOOTNOTES |
|---|
Received for publication 19 November 1997 and in final form 19 November 1997.
Address reprint requests to Dr. Norbert A. Dencher, Dept. Physical Biochemistry, Institute of Biochemistry, Darmstadt University of Technology, Petersenstr.22, D-64287 Darmstadt, Germany. Tel.: 49-6151-165275; Fax: 49-6151-164171; E-mail: dencher{at}pop.tu-darmstadt.de.
| |
REFERENCES |
|---|
Biophys J, January 1998, p. 1-2, Vol. 74, No. 1
© 1998 by the Biophysical Society 0006-3495/98/01/01/02 $2.00
This article has been cited by other articles:
 |
|
 |
|
  K. Scholtmeijer, M. I. Janssen, B. Gerssen, M. L. de Vocht, B. M. van Leeuwen, T. G. van Kooten, H. A. B. Wosten, and J. G. H. Wessels Surface Modifications Created by Using Engineered Hydrophobins Appl. Envir. Microbiol., March 1, 2002; 68(3): 1367 - 1373. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Wibbenmeyer, P. Schuck, S. J. Smith-Gill, and R. C. Willson Salt Links Dominate Affinity of Antibody HyHEL-5 for Lysozyme through Enthalpic Contributions J. Biol. Chem., September 17, 1999; 274(38): 26838 - 26842. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Shiroishi, A. Yokota, K. Tsumoto, H. Kondo, Y. Nishimiya, K. Horii, M. Matsushima, K. Ogasahara, K. Yutani, and I. Kumagai Structural Evidence for Entropic Contribution of Salt Bridge Formation to a Protein Antigen-Antibody Interaction. THE CASE OF HEN LYSOZYME-HyHEL-10 Fv COMPLEX J. Biol. Chem., June 15, 2001; 276(25): 23042 - 23050. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
