Matthew C. Blosser

Nucleobases bind to and stabilize aggregates of a prebiotic amphiphile, providing a viable mechanism for the emergence of protocells

Primordial cells presumably combined RNAs, which functioned as catalysts and carriers of genetic information, with an encapsulating membrane of aggregated amphiphilic molecules. Major questions regarding this hypothesis include how the four bases and the sugar in RNA were selected from a mixture of prebiotic compounds and colocalized with such membranes, and how the membranes were stabilized against flocculation in salt water. To address these questions, we explored the possibility that aggregates of decanoic acid, a prebiotic amphiphile, interact with the bases and sugar found in RNA. We found that these bases, as well as some but not all related bases, bind to decanoic acid aggregates. Moreover, both the bases and ribose inhibit flocculation of decanoic acid by salt. The extent of inhibition by the bases correlates with the extent of their binding, and ribose inhibits to a greater extent than three similar sugars. Finally, the stabilizing effects of a base and ribose are additive. Thus, aggregates of a prebiotic amphiphile bind certain heterocyclic bases and sugars, including those found in RNA, and this binding stabilizes the aggregates against salt. These mutually reinforcing mechanisms might have driven the emergence of protocells.

Transbilayer Colocalization of Lipid Domains Explained via Measurement of Strong Coupling Parameters

When micron-scale compositional heterogeneity develops in membranes, the distribution of lipids on one face of the membrane strongly affects the distribution on the other. Specifically, when lipid membranes phase separate into coexisting liquid phases, domains in each monolayer leaflet of the membrane are colocalized with domains in the opposite leaflet. Colocalized domains have never been observed to spontaneously move out of registry. This result indicates that the lipid compositions in one leaflet are strongly coupled to compositions in the opposing leaflet. Predictions of the interleaflet coupling parameter, Λ, vary by a factor of 50. We measure the value of Λ by applying high shear forces to supported lipid bilayers. This causes the upper leaflet to slide over the lower leaflet, moving domains out of registry. We find that the threshold shear stress required to deregister domains in the upper and lower leaflets increases with the inverse length of domains. We derive a simple, closed-form expression relating the threshold shear to Λ, and find Λ = 0.016 ± 0.004 kBT/nm2.

Minimal Effect of Lipid Charge on Membrane Miscibility Phase Behavior in Three Ternary Systems

Giant unilamellar vesicles composed of a ternary mixture of phospholipids and cholesterol exhibit coexisting liquid phases over a range of temperatures and compositions. A significant fraction of lipids in biological membranes are charged. Here, we present phase diagrams of vesicles composed of phosphatidylcholine (PC) lipids, which are zwitterionic; phosphatidylglycerol (PG) lipids, which are anionic; and cholesterol (Chol). Specifically, we use DiPhyPG-DPPC-Chol and DiPhyPC-DPPG-Chol. We show that miscibility in membranes containing charged PG lipids occurs over similarly high temperatures and broad lipid compositions as in corresponding membranes containing only uncharged lipids, and that the presence of salt has a minimal effect. We verified our results in two ways. First, we used mass spectrometry to ensure that charged PC/PG/Chol vesicles formed by gentle hydration have the same composition as the lipid stocks from which they are made. Second, we repeated the experiments by substituting phosphatidylserine for PG as the charged lipid and observed similar phenomena. Our results consistently support the view that monovalent charged lipids have only a minimal effect on lipid miscibility phase behavior in our system.

Thickness Mismatch of Coexisting Liquid Phases in Noncanonical Lipid Bilayers

Lipid composition dictates membrane thickness, which in turn can influence membrane protein activity. Lipid composition also determines whether a membrane demixes into coexisting liquid-crystalline phases. Previous direct measurements of demixed lipid membranes have always found a liquid-ordered phase that is thicker than the liquid-disordered phase. Here we investigated noncanonical ternary lipid mixtures designed to produce bilayers with thicker disordered phases than ordered phases. The membranes were composed of short, saturated (ordered) lipids; long, unsaturated (disordered) lipids; and cholesterol. We found that few of these systems yield coexisting liquid phases above 10 °C. For membranes that do demix into two liquid phases, we measured the thickness mismatch between the phases by atomic force microscopy and found that not one of the systems yields thicker disordered than ordered phases under standard experimental conditions. We found no monotonic relationship between demixing temperatures of these ternary systems and either estimated thickness mismatches between the liquid phases or the physical parameters of single-component membranes composed of the individual lipids. These results highlight the robustness of a membranes liquid-ordered phase to be thicker than the liquid-disordered phase, regardless of the membranes lipid composition.

A Self-Assembled Aggregate Composed of a Fatty Acid Membrane and the Building Blocks of Biological Polymers Provides a First Step in the Emergence of Protocells

We propose that the first step in the origin of cellular life on Earth was the self-assembly of fatty acids with the building blocks of RNA and protein, resulting in a stable aggregate. This scheme provides explanations for the selection and concentration of the prebiotic components of cells; the stabilization and growth of early membranes; the catalysis of biopolymer synthesis; and the co-localization of membranes, RNA and protein. In this article, we review the evidence and rationale for the formation of the proposed aggregate: (i) the well-established phenomenon of self-assembly of fatty acids to form vesicles; (ii) our published evidence that nucleobases and sugars bind to and stabilize such vesicles; and (iii) the reasons why amino acids likely do so as well. We then explain how the conformational constraints and altered chemical environment due to binding of the components to the membrane could facilitate the formation of nucleosides, oligonucleotides and peptides. We conclude by discussing how the resulting oligomers, even if short and random, could have increased vesicle stability and growth more than their building blocks did, and how competition among these vesicles could have led to longer polymers with complex functions.

Miscibility of Ternary Membranes Containing Charged Lipids and Confirmation of Membrane Composition by Mass Spectrometry

Here I present phase diagrams of vesicles containing phosphatidylcholine (PC), an uncharged lipid; phosphatidylglycerol (PG), a charged lipid; and cholesterol. I have found that this mixture exhibits coexisting liquid phases over a wide range of temperatures and compositions. I have found that miscibility in membranes containing charged lipids occurs over similar ranges of temperatures and lipid compositions as in membranes containing only uncharged lipids. Techniques for creating vesicle membranes containing charged lipids are significantly more challenging and less well characterized than for membranes containing neutral lipids. Here I use mass spectrometry to determine that the final membrane lipid composition is close to that of the initial stock solution. Specifically, I use a quadrupole spectrometer with an electrospray source using multiple reaction monitoring (MRM) in both positive and negative mode. Since the same instrument is used to quantify both the ratio of PC to PG and PC to cholesterol, the entire composition can be determined from one sample. The sensitivity of the technique is higher than phosphorus assays, and the MRM makes it extremely unlikely that signal is due to molecules other than the lipids of interest. This result confirms that vesicles made by gentle hydration have a predictable composition at both high and low fractions of charged lipids.

Miscibility Phase Behavior of GUV Membranes Containing Charge: Ternary Mixtures of Cholesterol, PC-Lipids, and PG-Lipids

Giant unilamellar vesicles composed of a ternary mixture of phospholipids and cholesterol exhibit coexisting liquid phases over a range of temperatures and compositions. Few studies of phase behavior have been made using charged lipids, even though they account for a significant fraction of lipids in biological membranes. Here, I present phase diagrams of vesicles containing phosphatidylcholine (PC), an uncharged lipid; phosphatidylglycerol (PG), a charged lipid; and cholesterol. These phase diagrams have several interesting features. Miscibility in membranes containing charged lipids occurs over similar ranges of temperatures and lipid compositions as in membranes containing only uncharged lipids. The coexisting liquid phases differ primarily in their phospholipid content such that one phase has a high concentration of charged lipid. Adding salt to the system causes an increase in transition temperatures at some membrane compositions, consistent with electrostatic screening, whereas the transition temperatures at other compositions fall.