For Biophysics Week, members of the Publications Committee selected a few influential articles from Biophysical Journal to highlight as well as the people who wrote them. This is the fourth article in the series.
Written by Publications Committee member William C. Wimley, Tulane University.
In 1774 the American polymath Benjamin Franklin had an opportunity to become the first membrane biophysicist (1, 2), but he let it slip away unnoticed. Franklin poured oil on a small pond in England to test its ability to calm waves. He wrote “The oil, though not more than a teaspoonful produced an instant calm over a space of several yards square, which spread amazingly…making all that quarter of the pond, perhaps half an acre, as smooth as a looking glass.” Franklin noted that the spreading oil pushed away objects that were floating on the surface of the water. He concluded “I think it is a curious enquiry, and I wish to know whence it arises.” (3) The concept of the molecule was more than a century old in 1774, yet neither Franklin nor any contemporary reasoned that he had created something like a molecular monolayer on the surface of the water. Thus, no one at the time made what would have been the first measurement of the thickness of a membrane (this would also have been the first measurement of the size of any molecule) — a teaspoonful (~5 ml) of oil spread over half an acre (~2000 m2) has a thickness of 2.5 nm, a value that is remarkably close to half of the ~5 nm thickness of a lipid bilayer membrane.
To celebrate the 60th anniversary of the Biophysical Journal, here I recount how, more than two centuries after Franklin stilled the waves on Clapham Common, Stephen White and postdocs Glen King and Michael Wiener used waves of another sort to describe how matter is distributed, and how thermal motions vary, across the thickness of the fluid phase lipid bilayer membrane. I discuss how this new image of the bilayer was acquired, how it transformed our understanding of bilayer structure, and how it catalyzed new insights in areas such as molecular dynamics simulations and membrane protein folding.
In the biologically relevant fluid phase, lipid bilayers are two dimensional fluids, not amenable to atomic resolution structure determination. The story of the “Complete Structure” is about the development of “liquid crystallography” used to extract maximum structural insight from available low-resolution lamellar X-ray and neutron diffraction. These authors published a series of seven papers, culminating in a seminal and highly cited 1992 Biophysical Journal paper “Structure of a Fluid Phase DOPC Bilayer by Joint Refinement of X-ray and Neutron Diffraction Data. III The Complete Structure” by Wiener and White (4). The first paper in the series, by King and White (5), established a framework for analyzing neutron (and X-ray) diffraction data using quasi-molecular groups; bonded groups of atoms expected to behave coherently. Examples of quasi-molecular groups include the choline headgroup, the glycerol backbone. and the terminal methyl groups. The second paper in the series, by Wiener and White, established a way to think about membrane diffraction (6). They showed that, in the absence of excess water, a stack of fluid phase bilayers is a near perfect one-dimensional crystal along the bilayer normal. The small number of diffraction orders observed, usually 4–8, is not due to stacking/lattice disorder, but to the inherent length-scale that best describes the system. The third paper in the series (7) established a “joint refinement” method in which the significant differences in X-ray and neutron atomic scattering cross-sections can be exploited to form a detailed image of the transbilayer distribution of quasi-molecular groups by global fitting of data sets acquired using both techniques. The fourth, fifth, and sixth papers (8-10) described the distribution of the fatty acid double bonds, terminal methyl groups and water. These papers also addressed the critical issue of scaling of neutron and X-ray diffraction data for the joint refinement. This body of work led to the “Complete Structure” paper in which the distribution of all lipid quasi-molecular groups and water were determined by a global fit of X-ray and neutron membrane diffraction scattering factors. The global fit was unconstrained except for the positions and widths of the water and double bonds which had been measured in the previous papers. The center of mass and 1/e Gaussian half-widths of each quasimolecular group were individually allowed to vary. The result, Fig. 1A, was the time averaged distribution (i.e., position and width) of each quasi-molecular group along the bilayer normal.
What did we learn from the “Complete Structure”?
The complete structure of a hydrated, fluid phase DOPC bilayer provided a holistic image of bilayer structure and dynamics, parts of which had previously been glimpsed by many other researchers (too many to name here). The fluid bilayer shows substantial thermal disorder in the transbilayer distributions of the lipid groups. Wiener and White showed that Gaussian distributions accurately describe the time-averaged positions of quasi-molecular groups along the bilayer normal. Comparison of the hard sphere widths with the experimentally determined widths showed that there are motional gradients. The glycerol backbone moiety has the lowest thermal motion, and thermal motion increases in both directions; toward the headgroup moieties and toward the acyl chains. The greatest thermal disorder occurs in the terminal methyl groups (11). A few years after the “Complete Structure,” Postdoc Kalina Hristova and White (12) made critical measurements of how bilayer thermal motions change as a function of hydration level, enabling the description of a more biologically relevant fluid phase bilayer in excess water.
Paradoxically, although the transbilayer Gaussian distributions of quasi-molecular groups are broad, their centers and widths are each determined with high precision in the “Complete Structure.” Thus, these experimentally determined transbilayer distributions of atomic groups can (and should!) be used to validate molecular dynamics simulations of lipid bilayers as White and colleagues have done (13). Experimentally validated (i.e., realistic) thermal motions (Figs. 1C and 1D) and subsequent lateral pressure profiles across the bilayer remain critical parameters for correctly modelling peptide/protein insertion, folding and structure in membrane simulations.
Another significant revelation in the “Complete Structure” was the true nature of the interface between the hydrocarbon core and the bulk water. Memorably described in the Complete Structure as a region of “tumultuous chemical heterogeneity,” the interfaces of a fluid bilayer occupy fully half the total thickness of the bilayer. All lipid groups, including the terminal methyl groups (11) spend some of their time in the interface due to their thermal motion. The interface also contains a significant amount of water. The time-averaged density of these groups creates a gradient of polarity, Fig. 1B, that forms a broad zone of transition between the very polar bulk water phase and the very non-polar hydrocarbon core, in the center of the bilayer. Importantly, the “Complete Structure” showed that the interfacial zones each occupy ~15Å along the bilayer normal, more than wide enough to encompass whole elements of protein secondary structure (Fig. 1B). Hristova, with White and others, demonstrated experimentally that amphipathic α-helical peptides are readily accommodated within the bilayer interfacial zone (14, 15).
The existence of these broad interfacial zones, with physical properties that are very different from the hydrocarbon core, means that a minimum of two hydrophobicity scales are needed to describe the thermodynamics of peptides and proteins partitioning into a lipid bilayer (16); at least one for the interface (17) and one for the hydrocarbon core (18). For peptides and proteins that partition into the interface, the reduced polarity, compared to bulk water, greatly increases the thermodynamic favorability of hydrogen bonded-secondary structure, giving rise to a very strong coupling between membrane binding and folding, a concept that has been useful in the understanding of membrane active peptides as well as membrane proteins (16).
Although the hydrocarbon core occupies only half the total thickness of the bilayer, the “Complete Structure” verified what many other researchers had previously concluded; the ~25-30 Å thick hydrocarbon core has a very low abundance of water and lipid polar groups, making it one of the most hydrophobic micro-environments known in biology. Gunnar von Heijne, with White and others later showed that the translocon, the protein machinery that folds and inserts membrane proteins in the endoplasmic reticulum, acts in accordance with the transmembrane distribution of polarity first revealed in the “Complete Structure” of DOPC (19). The effective hydrophobicity sensed by the amino acids in a potential membrane spanning helix is highest in the center of the membrane, becoming lower as the amino acid in question is moved away from the hydrocarbon core towards either interfacial zone. While the mechanistic details of translocon-mediated insertion and folding are still being revealed, the “Complete Structure” of DOPC set the stage for understanding the role of the bilayer physical properties in the process.
Conclusion
Although Benjamin Franklin missed his chance in 1774 to be the first membrane biophysicist, many other great scientists helped evolve our view of the structure of the lipid bilayer membrane (1, 20). For a long time, the prevailing image of a lipid bilayer had been that of a hard-edged slab of hydrocarbon, created by lipids with little or no thermal motion along the bilayer normal (such simplistic cartoons of bilayer structure also ignore the immense compositional complexity of real bilayers). Here, at the 60th anniversary of the Biophysical Journal, I have highlighted how Stephen White and colleagues contributed to our modern view of the bilayer by giving us the “Complete Structure” of a fluid phase lipid bilayer membrane. They enabled us to see the true dynamic complexity previously only glimpsed by many others. Unfortunately, old ideas, and simplistic cartoons die only very slowly. Incorrect and unrealistic depictions of static, rigid, uniform membrane structures, which may adversely affect how scientists and students think about membrane biology, have not yet been eradicated from textbooks, scientific papers, and the internet.
References
1. C. Tanford. 1989. Ben Franklin Stilled the Waves. An Informal History of Pouring Oil on Water with Reflections on the Ups and Downs of Scientific Life in General. Duke Univesity Press, Durham, NC.
2. D. N. Wang, H. Stieglitz, J. Marden, L. K. Tamm. 2013. Benjamin Franklin, Philadelphia's favorite son, was a membrane biophysicist. Biophys J. 104:287–291.
3. W. Brownrigg, M. Farish. 1774. Of the Stilling of Waves by means of Oil. Extracted from Sundry Letters between Benjamin Franklin, LL. D. F. R. S. William Brownrigg, M. D. F. R. S. and the Reverend Mr. Farish. Philosophical Transactions 64:445–460.
4. M. C. Wiener, S. H. White. 1992. Structure of a fluid dioleoylphosphatidylcholine bilayer determined by joint refinement of x-ray and neutron diffraction data. III. Complete structure. Biophys. J. 61:434–447.
5. G. I. King, S. H. White, 1986. Determining bilayer hydrocarbon thickness from neutron diffraction measurements using strip-function models. Biophys. J. 49:1047–1054.
6. M. C. Wiener, S. H. White. 1991. Fluid bilayer structure determination by the combined use of X-ray and neutron diffraction. I. Fluid bilayer models and the limits of resolution. Biophys. J. 59:162–173.
7. M. C. Wiener, S. H. White. 1991. Fluid bilayer structure determination by the combined use of X-ray and neutron diffraction. II. "Composition-space" refinement method. Biophys. J. 59: 174–185.
8. M. C. Wiener, S. H. White. 1991. Transbilayer distribution of bromine in fluid bilayers containing a specifically brominated analog of dioleoylphosphatidylcholine. Biochemistry 30: 6997–7008.
9. M. C. Wiener, G. I. King, S. H. White. 1991. Structure of a fluid dioleoylphosphatidylcholine bilayer determined by joint refinement of x-ray and neutron diffraction data. I. Scaling of neutron data and the distribution of double-bonds and water. Biophys. J. 60:568–576.
10. M. C. Wiener, S. H. White. 1992. Structure of a fluid dioleoylphosphatidylcholine bilayer determined by joint refinement of x-ray and neutron diffraction data. II. Distribution and packing of terminal methyl groups. Biophys. J. 61:428–433.
11. M. Mihailescu, R. G. Vaswani, E. Jardon-Valadez, F. Castro-Roman, J. A. Freites, D. L. Worcester, A. R. Chamberlin, D. J. Tobias, S. H. White. 2011. Acyl-chain methyl distributions of liquid-ordered and -disordered membranes. Biophys J. 100:1455–1462.
12. K. Hristova, S. H. White.1998. Determination of the hydrocarbon core structure of fluid dioleoylphosphocholine (DOPC) bilayers by x-ray diffraction using specific bromination of the double-bonds: Effect of hydration. Biophys. J. 74:2419–2433.
13. R. W. Benz, F. Castro-Roman, D. J. Tobias, S. H. White. 2005. Experimental validation of molecular dynamics simulations of lipid bilayers: a new approach. Biophys J. 88:805–817.
14. K. Hristova, C. E. Dempsey, S. H. White. 2001. Structure, location, and lipid perturbations of melittin at the membrane interface. Biophys. J. 80:801–811.
15. K. Hristova, W. C. Wimley, V. K. Mishra, G. M. Anantharamiah, J. P. Segrest, S. H. White. 1999. An amphipathic a-helix at a membrane interface: a structural study using a novel X-ray diffraction method. J. Mol. Biol. 290:99–117.
16. S. H. White, W. C. Wimley. 1999. Membrane protein folding and stability: physical principles. Annu. Rev. Biophys. Biomol. Struct. 28:319–365.
17. W. C. Wimley, S. H. White. 1996. Experimentally determined hydrophobicity scale for proteins at membrane interfaces. Nature Struct. Biol. 3:842–848.
18. W. C. Wimley, T. P. Creamer, S. H. White. 1996. Solvation energies of amino acid sidechains and backbone in a family of host-guest pentapeptides. Biochemistry 35:5109–5124.