The Functionality of Membrane-Inserting Proteins and Peptides: Curvature Sensing, Generation, and Pore Formation.

Proteins and peptides with hydrophobic and amphiphilic segments are responsible for many biological functions. The sensing and generation of membrane curvature are the functions of several protein domains or motifs. While some specific membrane proteins play an essential role in controlling the curvature of distinct intracellular membranes, others participate in various cellular processes such as clathrin-mediated endocytosis, where several proteins sort themselves at the neck of the membrane bud. A few membrane-inserting proteins form nanopores that permeate selective ions and water to cross the membrane. In addition, many natural and synthetic small peptides and protein toxins disrupt the membrane by inducing nonspecific pores in the membrane. The pore formation causes cell death through the uncontrolled exchange between interior and exterior cellular contents. In this article, we discuss the insertion depth and orientation of protein/peptide helices, and their role as a sensor and inducer of membrane curvature as well as a pore former in the membrane. We anticipate that this extensive review will assist biophysicists to gain insight into curvature sensing, generation, and pore formation by membrane insertion.

Insights into Membrane Curvature Sensing and Membrane Remodeling by Intrinsically Disordered Proteins and Protein Regions.

Cellular membranes are highly dynamic in shape. They can rapidly and precisely regulate their shape to perform various cellular functions. The protein's ability to sense membrane curvature is essential in various biological events such as cell signaling and membrane trafficking. As they are bound, these curvature-sensing proteins may also change the local membrane shape by one or more curvature driving mechanisms. Established curvature-sensing/driving mechanisms rely on proteins with specific structural features such as amphipathic helices and intrinsically curved shapes. However, the recent discovery and characterization of many proteins have shattered the protein structure-function paradigm, believing that the protein functions require a unique structural feature. Typically, such structure-independent functions are carried either entirely by intrinsically disordered proteins or hybrid proteins containing disordered regions and structured domains. It is becoming more apparent that disordered proteins and regions can be potent sensors/inducers of membrane curvatures. In this article, we outline the basic features of disordered proteins and regions, the motifs in such proteins that encode the function, membrane remodeling by disordered proteins and regions, and assays that may be employed to investigate curvature sensing and generation by ordered/disordered proteins.

Recent advancements to measure membrane mechanical and transport properties.

The natural vesicles, microscopic spherical structures defined by a single or many lipid bilayer membranes, not only entrap but are also dispersed in the aqueous environment. The space division between inner and outer compartments is also the basic characteristics of cell membranes playing several essential functions in all living organisms. Thus, vesicles are a simple model system for studying various cellular properties. In the last few decades, synthetic vesicles (or liposomes) have gained substantial popularity from many academia as model membranes and from many pharmaceutical industries as targeted and controlled drug delivery systems. The manufacturing of vesicles with desired characteristics that can entrap and release the drugs as required is one of the major challenges in this research area. To this end, a better understanding of the mechanical and transport properties of vesicles is essential to gain deeper insight into the fundamental biological mechanisms of vesicle formation and cellular uptake. The requirement has brought the modifications in membrane composition (with cholesterol, charged lipid, proteins, peptides, polymers, etc.) and solution conditions (with salts, pH, buffers, etc.). This article mainly focuses on the different techniques developed for studying the mechanical and transport properties of natural/synthetic vesicles. In particular, I thoroughly review the properties such as bending and stretching elastic moduli, lysis tension, and permeability of vesicle membranes.

Recent developments in membrane curvature sensing and induction by proteins.

BACKGROUND: Membrane-bound intracellular organelles have characteristic shapes attributed to different local membrane curvatures, and these attributes are conserved across species. Over the past decade, it has been confirmed that specific proteins control the large curvatures of the membrane, whereas many others due to their specific structural features can sense the curvatures and bind to the specific geometrical cues. Elucidating the interplay between sensing and induction is indispensable to understand the mechanisms behind various biological processes such as vesicular trafficking and budding. SCOPE OF REVIEW: We provide an overview of major classes of membrane proteins and the mechanisms of curvature sensing and induction. We then discuss the importance of membrane elastic characteristics to induce the membrane shapes similar to intracellular organelles. Finally, we survey recently available assays developed for studying the curvature sensing and induction by many proteins. MAJOR CONCLUSIONS: Recent theoretical/computational modeling along with experimental studies have uncovered fascinating connections between lipid membrane and protein interactions. However, the phenomena of protein localization and synchronization to generate spatiotemporal dynamics in membrane morphology are yet to be fully understood. GENERAL SIGNIFICANCE: The understanding of protein-membrane interactions is essential to shed light on various biological processes. This further enables the technological applications of many natural proteins/peptides in therapeutic treatments. The studies of membrane dynamic shapes help to understand the fundamental functions of membranes, while the medicinal roles of various macromolecules (such as proteins, peptides, etc.) are being increasingly investigated.

Vesicle formation mechanisms: an overview.

Vesicle structures primarily embody spherical capsules composed of a single or multiple bilayers, entrapping a pool of aqueous solution in their interior. The bilayers can be synthesised by phospholipids or other amphiphiles (surfactants, block copolymers, etc.). Vesicles with broad-spectrum applications in numerous scientific disciplines, including biochemistry, biophysics, biology, and various pharmaceutical industries, have attracted widespread attention. Consequently, a multitude of protocols have been devised and proposed for their fabrication. In this review, with a motivation to derive the basic conditions for the formation of vesicles, the associated thermodynamic and kinetic aspects are comprehensively appraised. Contextually, an all-purpose overview of the underlying thermodynamics of bilayer/membrane generation and deformation, including the chemical potential of aggregates, geometric packing and the concept of elastic properties, is presented. Additionally, the current review highlights the probable, inherent mechanisms of vesicle formation under distinct modes of manufacturing. We lay focus on vesicle formation from pre-existing bilayers, as well as from bilayers, which form when lipids from an organic solvent are transferred into an aqueous medium. Furthermore, we outline the kinetic effects on vesicle formation from the lamellar phase, with and without the presence of shearing force. Wherever required, the experimental and/or theoretical outcomes, the driving forces for vesicle size selection, and various scaling laws are also reviewed, all of which facilitate an overall improved understanding of the vesicle formation mechanisms.

Rapid single-step formation of liposomes by flow assisted stationary phase interdiffusion.

Laboratory preparation of unilamellar liposomes often involves multiple steps carried out over several hours to achieve a monodisperse size distribution. Here, we present a methodology based on a recently introduced lipid self-assembly principle-stationary phase interdiffusion (SPI)-to prepare large unilamellar vesicles (LUVs) of a monodisperse population in a short period of about 10 min. The stationary interface between a lipid-ethanol phase and an aqueous phase is created by a density difference induced convective flow in a horizontal capillary. The average size of the liposomes, as expected from the SPI principle, is modulated only by the temperature and the type of lipids. Lipid concentration, ethanol content, pH of the aqueous phase, and the time duration of the experiment have little influence on the mean diameter of the vesicles. This simple methodology can be easily carried out with a capillary and a micro-needled syringe and provides a rapid production tool for researchers requiring reproducible liposome suspensions. Refined natural lipids, based on soy and egg lecithin mixtures, yield LUVs in the range 100-200 nm, suitable for drug delivery applications.

Spontaneous formation of single component liposomes from a solution.

The diameter of lipid vesicles is generally known to be determined by parameters external to the system, such as fluid shear, electric fields, co-surfactants, etc. We present a mechanism by which a system consisting of a single component lipid can spontaneously assemble from a solution phase to form monodisperse unilamellar vesicles of well-defined diameters dictated only by thermodynamic parameters intrinsic to the system. Here, the lipids self-assemble as vesicles when an aqueous phase diffusively replaces the original solvent in a macroscopically stationary (or quiescent) manner. We demonstrate this using phosphatidyl choline lipid-ethanol-water systems, where the average diameter of the liposomes is shown to be intrinsic, in reasonable agreement with the Helfrich's model of the vesicle free energy. The size depends only on the temperature and the lipid type, eliminating dependence on kinetic effects or external forcing normally observed. The method provides the first pure system to study the self-assembly of vesicle-forming surfactants; and with a natural thermodynamic length scale, it may have an implication for the vesicle size selection under pre-biotic conditions.