GENERAL IDEA

Here we discuss the conceptual foundations of single-molecule biophysics in the context of cellular biology. We provide an overview of existing ensemble average techniques used to study biological processes and consider the importance of single-molecule biology experiments.

Introduction

Some of the most talented physicists in modern science history have been led ultimately to address challenging questions of biology. This is exemplified in Erwin Schrödinger’s essay ‘What is Life?’ (see Schrödinger, 1944). It starts with the question ‘How can the events in space and time which take place within the spatial boundary of a living organism be accounted for by physics and chemistry?’ In other words, can we address the big questions of the life sciences from the standpoint of the physical sciences. The ~60 years following the publication of this seminal work has seen a vast increase in our understanding of biology at the molecular scale, and the physical sciences have played a key role in resolving many central problems. The biological problems have not been made easier by the absence of a compelling and consistent definition of ‘life’ – writing from the context for a meaning of ‘artificial life’, the American journalist Steven Levy noted 48 examples of definitions of life from eminent scientists, no two of which were the same (Levy, 1993). From a physical perspective, life is a means of trapping free energy (ultimately from the sun or, more rarely, geological thermal vents) into units of increased local order, which in effect locally decrease entropy, while the units maintain their status in situations which are generally far from thermal equilibrium.

GENERAL IDEA

Here we explore some of the pioneering single-molecule experiments that have increased our understanding of the processes that essentially involve foreign molecules external to cells either binding to the cell surface or being internalized by cells. Such molecules interact directly with the semipermeable membrane of the cell, making it an exceptionally lively and dynamic environment.

Introduction

Highly efficient mechanisms exist that allow foreign molecules to bind to cell surfaces, ultimately evoking some form of signal response, and allow a variety of external molecules over a broad range of size, chemistry and charge to enter cells. At one level these mechanisms include processes involving receptor molecules embedded in the outer membrane of cells that bind to ligand molecules. Many of the stages of this detection and signal transduction process have canonical features, sometimes involving adapter molecules binding to the original ligand, as well as specific binding events and changes in molecular conformation which can often be transmitted over relatively long length scales of several nanometres spanning one or sometimes more lipid bilayer membranes and sometimes involving cooperative effects from other receptor molecules. Non-native particles which are internalized by cells range in size from single molecules to much larger heterogenous macromolecular complexes such as viruses. In this chapter we will encounter first-hand examples of how these processes have been investigated using single-molecule biophysics. These investigations reveal highly complex behaviours, indicating that the world outside the cell is just as important as the world on the inside.

GENERAL IDEA

Here we introduce the seminal biophysics investigations which have transformed our understanding of biology at the single-molecule level, and lay the foundations for describing single-biomolecule experimentation on functioning live cells

Introduction

An instructive exercise for those learning about single-molecule biophysics is to compile a list of one’s own top ten research papers of all time. The choice is obviously subject to a great deal of personal bias, and is a dynamic structure which may change with time, and some of the early, seminal papers on the list might later be superseded by subsequent incarnations with more of the original unresolved questions resolved (and maybe even with the ‘right’ answers, as opposed to what were perhaps novel but slightly incorrect ‘best guesses’ at the time!). Even so, as a process for understanding how, and why, single-molecule biophysics has evolved the way it has, and how it is likely to progress into the arena of far greater physiological relevance in the near future, the reader might find the exercise suprisingly fulfilling. In this chapter we discuss some of the strong candidates for this list of seminal papers, and lay the foundations for the remaining chapters in this book which describe real single-biomolecule experiments performed either on living cells or in an environment which has substantial physiological relevance.

GENERAL IDEA

In this chapter we encounter some key examples of single molecules and molecular complexes which are primarily integrated into the cell membrane performing a range of essential biological functions, and of the surrounding molecular architecture of the phospholipids, that have been studied extensively using exemplary single-molecule biophysics techniques

Introduction

Roughly 30% of all proteins are integrated into the membranes of cells, a significant proportion which is indicative of the importance of molecular processes which occur at the surfaces of cells. The cell membrane is an enormously important structure. It provides a physical support for seeding a vast array of complex surface chemistry reactions, as well as acting as the site for molecular detectors, pumps, channels and motors, not to mention its obvious function as a physical boundary to the cell. In the previous chapter, we discussed some of the important biological systems that deal with molecules and molecular complexes which spend significant periods outside the cell membrane boundary, and how single-molecule methods have dramatically improved our understanding of these processes. In this chapter, we will discuss several of the biological systems which are primarily associated directly with the cell membrane itself, and how single-molecule techniques have probed many of these in physiologically relevant settings, including both protein complexes integrated into the membrane and the makeup of the phosopholipid bilayer. These include molecular complexes which transport molecules across cell membranes, such as ion channels and protein transport nanopores, as well as some remarkable molecular machines which are involved in cell motility and cellular fuel manufacture.

Life, from the bottom up

Richard Feynman, celebrated physicist, science communicator and bongo-drum enthusiast, gave a lecture in Caltech, USA, a few days after Christmas 1959, that would come to be seen by future nanotechnologists as essentially prophetic. His talk was entitled ‘There’s plenty of room at the bottom’, and was concerned primarily with discussing the feasibility of a future ability to store information and to control and manipulate machines on a length scale which was tens of thousands of times smaller than that of the macroscopic world of things like typical books and electric motors of that day. It was essentially a clarion call to scientists and engineers to develop a new field, which would later be termed nanotechnology (see Taniguchi, 1974). But in one aside, Feynman alluded to the very small scale of biological systems, and how cells used these to do ‘all kinds of marvelous things’, which in its own small way has been wisely prescient for the subsequent seismic shifts in our understanding of how biological systems really work. We now know that the fundamental minimal functional unit which can adequately describe the properties of these systems is the single biological molecule. That is not to say that the constituent atoms at smaller length scales do not matter, nor the sub-atomic particles that make up the individual atoms, nor smaller still the quarks that make up the sub-atomic particles. Rather that, in general, we do not need to refer to a length scale smaller than the single molecule to understand biological processes.