squishy proteins in fluid membranes
TRANSCRIPT
COMMUNICATION
Squishy proteins in fluid membranes
MYER BLOOM Dc,pnr~r~letll ($Physics, Utriuersily ofBrilislt Collr~~lbirr, Vrrtlco~rcier, B .C. , Crrtzrrrirl V6T i W 5
Received September 25, 1979
A variety of experiments indicate that proteins hardly perturb the orientational order of phospholipids in biological membranes. It is suggested that, in common with soluble proteins, integral proteins in membranes have a "fluid-like" outer region which provides an approximate fluid mechanical match with the liquid crystalline phospholipid membrane.
Diverses experiences indiquent que les proteines ne perturbent presque pas I'ordre orien- tationnel des phospholipides dans les membranes biologiques. On propose comme explication que les proteines enti t res dans les membranes possedent, en commun avec les proteines solubles, une region externe "fluidiforme" qui permet une jonction mecanique approximativement fluide avec le cristal liquide d e phospholipides d e la membrane.
Can. J. Phys., 57. 2227 (1979) [Traduit par le journal]
Little information is available on the structure of those proteins which are integrated with mem- branes. The use of diffraction methods has been limited thus far to a single case (1) because of the lack of translational symmetry in the fluid state of biological membranes in which most biological ac- tivity takes place (2, 3) and the difficulty of crystal- lizing membrane-bound proteins. A related prob- lem, for which no definitive solution has yet been found, concerns the nature of the interaction be- tween phospholipid and protein molecules which is responsible for the influence of the physical state of the membrane on the function of the proteins. However, the physical nature of membrane fluidity and the most fundamental properties of the fluid phase of phospholipid bilayer model membranes have been well known for several years now. The main contribution to changes in the thermodynamic properties of membranes which undergo gel-to- liquid crystalline phase transitions is associated with chain melting, i.e., increased orientational disorder of the C-C bonds of the fatty acyl chains of the phospholipid molecules (3). Measurements using deuterium nuclear magnetic resonance (4, 5) ('H-nmr) have provided a microscopic measure of the local orientational order of the acyl chains, so that the characteristic "fluid bilayer signature" of the variation of orientational order with depth in model membranes is now available. The corre- sponding results for the physical properties of the phospholipid component of bacterial membranes (6- 10) and reconstituted membranes containing a single type of protein (1 1-13) are just now becom- ing available. In addition, an elegant experiment to
explore selectively the orientational order of the acyl chains in the immediate vicinity of rhodopsin using electron paramagnetic resonance (epr) spin label fatty acids covalently attached to this protein has recently been carrried out (14, 15).
The purpose of this communication is to point out that the available data suggest strongly that the external parts of the integral membrane proteins, i.e., those regions in contact with the membrane phospholipids, are also fluid-like, undergoing sub- stantial angular fluctuations on a time scale of the order of lop5 s. This suggestion is consistent with theoretical investigations of internal motions in proteins using molecular dynamics computer methods (16), with the implications of recent X-ray diffraction studies of water-soluble proteins in crystals (17, 18) and with high resolution nmr studies of side chain motions in proteins in aqueous solutions (19). It implies that a major reason that most biological activity of membrane-bound pro- teins takes place at temperatures sufficiently high that most of the membrane phospholipids are in their liquid crystalline phase is that the fluid phase allows the proteins to undergo the large amplitude internal motions required for enzymatic activity.
The variation with depth of the "orientational order parameter" S of the acyl chains in the liquid crystalline phase of model membranes is influenced by the anisotropic intermolecular interactions be- tween each phospholipid molecule and its neighbors and has been found to be practically in- dependent of the polar head group of the phos- pholipid molecule, providing that the different bilayer systems are compared at the same "re-
0008-4204/79/122227-04$0 1. 00/0 @ 1979 National Research Council of Canada/Conseil national d e recherches du Canada
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duced temperature" t = T - T,, where T, is the gel-to-liquid crystalline transition temperature (20). The difference between the sn- 1 and sn-2 chains in a saturated phospholipid molecule can be explained in terms of constraints on the different orientations of the C-C bonds with respect to the glycerol backbone (5, 20). while the differences between saturated and unsaturated chains are due primarily to the geometrical effects of the double bond (21). The results of 'H-nmr measurements of the mem- branes of Acholeplasn~a laidla~vii and Eschet-ichia coli bacteria are now available for a large number of methylene group positions in both saturated (6) and unsaturated (9) hydrocarbon chains. The re- sults are remarkable. In spite of the fact that the percentage (by weight) of protein in these bacterial systems is well over 50%, the magnitudes of the orientational order parameters of the methylene deuterons in the fluid regions are either slightly smaller or differ very little from those found in model membranes for every one of the many posi- tions studied thus far on both the saturated chains (6, 20) and the unsaturated chains (9, 21) (and per- sonal communications by K. R. Jeffrey, J . Seelig, and I. C. P. Smith). The same result has been found in %-nmr studies of reconstituted membranes in- volving various integral proteins in different phos- pholipid bilayer systems (12, 13) and for epr spin label experiments (14, 15) on hydrocarbon chains attached covalently to rhodopsin molecules in re- constituted membranes composed of DMPC and DPPC phospholipid molecules.
Though one may argue that very little informa- tion is available on the fraction of the total protein mass which is actually inside the hydrophobic re- gions of the bacterial membranes, it is now known from electron diffraction studies (R. Henderson, personal communication) that this fraction is ap- proximately 40% for cytochrome c oxidase, the best studied integral protein in reconstituted mem- branes using 'H-nmr methods (I I- 13). Therefore, for the largest concentrations of cytochrome c oxidase in reconstituted membranes studied using 'H-nmr, the phospholipids were in direct contact with the enzyme most of the time. In the case of the epr studies (14, 15), the spin label selectively probes the phospholipids close to the rhodopsin molecules, which are themselves substantially within the bilayer (22). Yet, no appreciable pertur- bation to the acyl chains was found in either case. What does this negative result mean? Are the in- teractions between lipids and proteins so weak that replacing more than 50% of a phospholipid bilayer by proteins has no influence on the orientational order of the acyl chains of the remaining phos- pholipid molecules?
A theoretical answer has been given by Marcelja (23). He showed that the anisotropic van del- Waals interactions between a rigid hydrophobic wall (which is Marcelja's model for proteins inside bilayers) should cause a substantial increase in the orientational order of the acyl chains of lipid mole- cules next to the wall. From an experimental standpoint, it is known that cholesterol in mem- branes does give rise to a substantial stiffening of those parts of the hydrocarbon chains in contact with the rigid section of the cholesterol molecules (24, 25).
Seelig and Seelig (I 3) and Oldfield et al. (12) have each independently ascribed the orientational dis- ordering effect of the lipid-protein interaction on the acyl chains to the roughness of the protein-lipid interface. They point out (personal communica- tions by E. Oldfield and J . Seelig) that plausible molecular models of the protein indicate the exis- tence of a rough protein surface at the molecular level due to amino acid side chains projecting at odd angles from common secondary structures such as a-helices. Motion of the phospholipid molecules along this rough protein surface at a rate very fast compared with the 'H-nmr averaging time of about
s could then give the required reduction in orientational order.
We propose here an alternative mechanism of orientational disordering at the lipid-protein interface. The basis of our proposal is that, as a result of the many internal degrees of freedom of the protein molecule, the protein surface is capable of undergoing substantial spatial displacements. Motions of individual amino acid side chains near the surface can give rise to large amplitude, localized density fluctuations, while collective mo- tions of the large number of atomic position vari- ables can generate wave-like motions of the protein surface. Such motions have been invoked in the interpretation of the results of recent X-ray diffrac- tion studies of crystals of water-soluble proteins (17, 18) and are consistent with molecular dynamics calculations of atomic motions in proteins by Karplus and his co-workers (16). The inner core of protein molecules in solution has been described as being "solid-like" and the outer regions as "fluid- like" in their motions. High resolution nmr experi- ments (19) indicate that though the average struc- tures of proteins in solution are usually no different from those in the protein crystals, the amplitude of the thermal motions tends to be greater for the proteins in solution.
If the density fluctuations and surface waves of integral membrane proteins are also "fluid-like", they could match the corresponding fluctuations in the fluid phase of the acyl chains of phospholipid
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COMMUNICATION 2229
bilayer systems and thereby explain, in a unified and natural manner, the 'H-nmr and epr spin label orientational order data described above. Although it is not possible, on the basis of presently available experimental data, to distinguish this dynamical lipid-protein interface model from the static model discussed above, it should be possible to design experiments to do so, though not using the power- ful diffraction methods available for the study of crystals formed by soluble proteins. For example, preliminary experiments of the 'H-nmr spin-lattice and spin-spin relaxation times in reconstituted membranes (12, 13) indicate that the protein acts to slow down the chain reorientational motions somewhat. Similar results have been obtained in 'H-nmr studies (26). It should be possible to study separately the spectral density of orientational fluctuations in proteins and lipids using a reconsti- tuted membrane with a single type of protein in a completely deuterated phospholipid membrane. By measuring the relaxation times of the proton spins in the proteins and the deuterium spins in the phospholipid molecules over a wide frequency range, a tedious and lengthy but by no means im- possible experiment, it may be possible to check on whether the orientational fluctuations of pro- tein-lipid interface manifest themselves in the mo- tions of each of the components.
One of the interesting aspects of the lipid-protein interaction is that it can give rise to an effective protein-protein interaction. A rigid protein-lipid surface has been shown, using two different forms of the lipid-protein interaction (23, 27), to produce a short range attractive force between proteins which could give rise to protein aggregation. Schroeder (28) has shown that an attractive effec- tive protein-protein interaction is a general conse- quence of any lipid-protein interaction which in- creases the orientational order of the hydrocarbon chains of the lipid molecules. It would be of interest to generalize the lipid-protein boundary conditions in studies of this type to include the possibility of an interface whose shape is modulated at a non-zero
trigger an attractive interaction between specific pairs of proteins. Other better established enzyma- tic functions associated with internal motions of proteins have been discussed in some of the papers already cited (16- 19).
In summary, we have presented a plausible in- terpretation of the observations, in a variety of biological and reconstituted membranes, that the proteins have not perturbed the orientational order of the hydrocarbon chains appreciably. Our con- jecture, that the surfaces of integral proteins in membranes tend to be fluidlike and thereby provide an improved fluid mechanical match to the liquid crystalline nature of the phospholipid molecules, is capable of being tested experimentally. A specific experiment involving nmr methods has been sug- gested for this purpose but it is hoped that this communication will stimulate experiments using other techniques.
Acknowledgments This paper was written while participating in the
summer school on Biophysics of Membranes and Intercellular Communication held in August 1979 at Les Houches, France. I am grateful to the many students and faculty of this school, and especially J . Davoust and P. Devaux, who made helpful and encouragingsuggestions. I thankc . C. F. Blake, D. Chapman, A. G. Redfield, R. J . P. Williams, and M. Zukermann for helpful discussions. In addition, I thank H . Frauenfelder for sending me the preprint which stimulated this work, J . Charvolin for his hospitality during my sabbatical leave at the Laboratoire de Physique des Solides, Orsay, France, where much of this work was carried out, the Killam Foundation for the award of an Izaak Walton Killam Memorial Scholarship, and the Na- tional Research Council of Canada for their con- tinuing support of my research program.
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