Exactly one year ago today I defended my doctoral thesis in Delft. Fast-forward one year, and I find myself writing these words in the California sun. How different life has become! Having embarked on an exciting new project, I am now challenged to think about a whole new universe of fascinating problems. Yet I do enjoy looking back every now and then to remember the good times of grad school. The good times of a protein in a box.
What is life? At the very least it is a concept that we humans find surprisingly difficult to define. Though generally wet and dynamic1, arguments about what defines life inevitably involve terms like ‘reproduction’, ‘growth’, and ‘adaptation’ – matters very common to cells and viruses alike, yet whether the latter belong to the category of living things is a matter of ongoing debate. For the purpose of this thesis (as well as for my own understanding), I adopt a less materialistic and more conceptual definition of life:
Life is a process brought forward by the self-organization of molecules, a process that seemingly violates the second law of thermodynamics2 as it increasingly acquires and maintains information over timescales that vastly exceed the lifetime of the molecules holding this information.
In this manner the distinction between a virus and a cell becomes rather meaningless: viruses are just as much part of the process that we know as ‘life’ as a homo sapiens like you or me is.3 In addition, it allows me to conveniently classify my thesis work as “an effort to gain better understanding of the molecular processes and building blocks of life”. Though this classification is rather broad – as myriads of doctoral theses written over the past century or so belong to this category – my thesis belongs to a relatively small and novel subcategory of the ‘gaining insight into the building blocks of life’ class by making use of two concepts: single-molecule and bottom-up approach.
Gaining direct, real-time access to the behavior of single molecules was a great leap forward a couple of decades ago. Not only did direct access to the reaction kinetics of individual molecules prove to provide crucial insights into the stochastic behavior and transient dynamics unavoidably obscured in the ensemble-averaged outcomes of classical bulk experiments, the idea of watching an individual, nanometer-sized DNA polymerase performing life’s work was simply fascinating. What made this possible to a large extent, was stray physicists wandering into the field of biology with in mind the idealized system of a particle in a box. Credit to the idea of starting from the bottom upwards with an as simple as possible system – a protein in a box – to study and quantify the fundamentals of life goes to the experimental physicists pioneering this field.
Anno 2016, watching a single molecule in action is still fascinating, yet no longer a truly remarkable achievement. The ability to observe and manipulate single proteins, multienzyme complexes, or cells has become commonplace, and even accessible by light microscopy4 through applying some clever tricks. But what do we learn from observing all these single events? Have single-molecule techniques become invaluable to the field of biology, fundamentally changing the way we think about life, like e.g. the discovery of the DNA double helix structure by Watson, Crick, and Franklin has done? Or is the field doomed to have an impact of lesser greatness: sexy, yet convicted to an existence in the shadows of the true fundamental insights that shaped the way we think about life – insights gained by the more traditional fields of biology, physics and chemistry?
Personally, I would say it is still too early to tell, but that the techniques developed in the field of single-molecule biophysics have proven useful in innumerable ways. Single-molecule experiments have the potential (and are well on their way) to become a tool used not only by scientists with a background in applied physics or engineering, but also by biologists – i.e. the scientists who generally have a better idea of which biology-related questions to ask. While knowing the exact behavior of single molecules is generally not pivotal to understanding entropic (i.e. non-living) systems, this does not hold true for the entropy-defying building blocks of life. However, as a result of the very different schools of thought that underlie the disciplines of biophysics and biology, both communities tend to communicate at their own specific wavelength – maybe keeping single-molecule biophysics more at the periphery of biology than it should be. Though there is definitely a trend towards increased interdisciplinarity, there is still sometimes a reciprocal lack of appreciation as a result.
With this in mind, l like to take the liberty of placing my thesis – also the result of the collective effort of the many great scientists I had the pleasure to learn from and work with over the past years – in a sub-subcategory by employing and developing an additional concept: multiplexed force spectroscopy. As the ensuing chapters will elaborate on, there is a huge gap between the behavior of a single molecule and the behavior of an ensemble of billions of molecules. Observing single events of protein binding or enzymatic complex activity makes poor statistics, especially if it is the rare, out of the ordinary behavior that is of interest. Repetition is key: crucial to fully appreciate the richness of detail that single-molecule observations add to the known ensemble-averaged behavior. Large scale repetition in parallel – multiplexing – makes it possible to gain these insights on such timescales that they can actually contribute to a successful career in science as well.
Gaining quantitative insights from single or rare events, while being able to convincingly place these in the bigger picture of their biological framework, is what multiplexed single-molecule force spectroscopy in essence aims to achieve. Multiplexed single-molecule force spectroscopy, in my opinion, can be regarded as a tool to bridge the gap between the single and the collective behavior of life, a tool to further enhance the contribution of the field of biophysics to understanding the processes of life. I hope this is what the careful reader will be able to appreciate throughout this thesis.
Delft, April 2016
Published as the preface to my doctoral thesis, April 5 2016. To paraphrase Howard C. Berg in ‘Random walks in biology’.
 The second law of thermodynamics states that, to quote Max Planck: “Every process occurring in nature
proceeds in the sense in which the sum of the entropies of all bodies taking part in the process is increased.”
In other words: the amount of randomness or disorder must always increase.
 This definition might invoke cognitive dissonances for future generations that might co-exist with artificial
 Though super-resolution microscopy of living cells is typically a top-down approach