Introduction - Diffusion of Lipids and Proteins in Complex Membranes

1. INTRODUCTION

Cellular membranes are utterly complex quasi-two-dimensional soft sheets of lipids that host a plethora of macromolecules such as membrane proteins and carbohydrates.

These membranes encapsulate numerous organelles — including the nucleus — in the cytoplasm. Perhaps the most central of all cellular membranes is the plasma membrane, which separates this intracellular environment of a cell from its surround-ings, hence regulating the transport of matter and messages to and from the cell [1].

The lipid bilayer — the main building block of cellular membranes — is made up of thousands of diﬀerent types of lipids that are distributed unevenly both along the bilayer plane as well as across the two bilayer leaflets [2, 3]. Furthermore, the plasma membrane is also tremendously crowded by thousands of diﬀerent kinds of membrane proteins — the small factories that are responsible for numerous cellular functions, such as signaling and transport [4, 5, 6]. The correct functioning of these proteins is of utmost importance for health, which is highlighted by the fact that a significant fraction of modern pharmaceuticals targets them [5].

In addition to being structurally complex, the plasma membrane is highly dynamic as its components are under constant motion driven by both thermal fluctuations and active transport processes [1]. Lipids and proteins diﬀuse along the membrane to form functional protein oligomers [7], lipid nanodomains [8], and to engage in specific lipid–protein interactions [9] that provide proteins with suitable environments to carry out their functions. These dynamic processes are certainly aﬀected or perhaps even regulated by the complexity characteristic of biomembranes, yet the details of this structure–dynamics–function interplay have remained poorly understood to date.

Experimental eﬀorts aiming to understand membrane dynamics have several lim-itations. Measurements of the dynamics in living cells suﬀer from rather poor spatiotemporal resolution as well as from the lack of proper control experiments.

Model membranes allow for a more controlled and systematic approach and there-fore provide a slight improvement in the obtained temporal and spatial resolutions.

2 1. Introduction Unfortunately, such model systems lack the proper non-equilibrium conditions that define living organisms.

Fortunately, experiments are not facing the aforementioned challenges alone. The-oreticians have studied dynamic processes for decades, and the field is currently as active as ever. Most importantly, molecular dynamics simulations of biomem-branes have reached their golden era in the recent years. Notably, the simulation field has seen substantial improvements in both the accuracy of the used models as well as the time and length scales reachable by the ever improving multiscale simulation approaches and the increasing computing capacity. These scales are currently already overlapping with those achieved by the most precise experimental methods. Simulations can hence act as the “ultimate microscope” and complement experiments by providing one with a nanoscale picture of the studied phenomena.

Such a multidisciplinary approach will certainly help us understand the dynamic processes taking place in complex membranes and therefore open new avenues to improve our health. In these eﬀorts, computer simulations — such as those employed in this Thesis — will undoubtedly be an indispensable tool.

Research Objectives and the Scope of This Thesis

The motion of lipids and proteins in biomembranes is traditionally described by empirical parameters, such as the diﬀusion coeﬃcient and the diﬀusion exponent.

The diﬀusion coeﬃcient describes the rate of diﬀusion, whereas the diﬀusion exponent distinguishes normal diﬀusion from its anomalous counterparts. Surprisingly little is actually known on how these two parameters depend on the structural complexities present in the plasma membrane. Moreover, the biological role of anomalous diﬀusion in membrane-associated processes has remained a mystery.

This Thesis has three central objectives related to improving our understanding of membrane dynamics. The first set of goals considers models that are commonly employed to predict lipid and protein diﬀusion coeﬃcients. The free area model for lipid bilayers [10] assumes that lipids diﬀuse via jumps between vacant sites in a membrane. It provides the diﬀusion coeﬃcient of a lipid as a function of two parameters — one describing the energy required for the lipid to break free from its old environment and the other related to the free area required for a jump.

This model has successfully been adapted to lipid bilayers [10], even though the underlying mechanism has been questioned [11]. We put the free area model to the

3 test in lipid monolayers, where the area parameter can be readily varied. Here, the simulation approach provided a straightforward and self-consistent evaluation, since the parameters obtained by fitting the free area model to lipid diﬀusion coeﬃcients can be readily compared to the corresponding parameters extracted directly from the simulations. The central question here was whether the parameters provided by the free area model to describe lipid motion are physically reasonable. If not so, the model is likely unable to capture the correct physical mechanism of lipid diﬀusion in monolayers, and hence its predictive power needs to be seriously questioned therein.

The Saﬀman–Delbrück model [12, 13] links protein diﬀusion coeﬃcient to parameters describing the protein, the membrane, and the surrounding solvent. Notably, it suggests a weak logarithmic dependence between the protein diﬀusion coeﬃcient and its radius. The Saﬀman–Delbrück model was derived for a single protein diﬀusing in an indefinitely large membrane sheet. However, the plasma membrane is exceptionally crowded with proteins [4], and it is, therefore, possible that the predictions of the model fail in such a crowded setting. We evaluated the ability of the Saﬀman–

Delbrück model to describe the size dependence of protein diﬀusion in crowded membranes. The main question here was whether the model is valid under crowding and, if not, what replaces it in such a setting.

Our second set of objectives considers lipid and protein dynamics in crowded mem-branes, as well as lipid dynamics in packed monolayers. It is known that crowding slows down diﬀusion [14] and induces anomalous diﬀusion [15]. However, the details of anomalous diﬀusion, especially the underlying physical mechanisms, have remained unsolved. We studied lipid and protein motion in membranes with diﬀerent levels of crowding. Here, we presented multiple research questions. What is the time regime in which anomalous diﬀusion manifests itself? How does the diﬀusion exponent vary as a function of lag time? Furthermore, how are these two related to the level of protein crowding? Similar questions were also tackled with the monolayer simulations, yet instead of protein crowding, here we systematically varied lipid packing. Moreover, we asked which mathematical model can describe anomalous subdiﬀusion in protein-crowded membranes: does the fractional Brownian motion — validated as the corresponding mechanism in protein-free membranes by us [16] — also hold in crowded conditions and, if not, what is the correct formulation.

Our third set of objectives considers methodology. Here, we aimed to provide an improved approach for embedding proteins in lipid membranes to foster studies on membrane protein systems. Moreover, we set an aim to improve the ability of

4 1. Introduction a commonly used simulation model to describe protein–protein interactions and hence provide reliable results for the dynamics of membranes crowded by proteins.

We consider that both of these improvements were crucial for reaching the other objectives described above.

Finally, the grand aim of this Thesis is to combine my work and the work of others into a comprehensive state-of-the-art picture of the current understanding of lipid and protein diﬀusion in complex biomembranes.

Contents of This Thesis

After this Introduction, being Chapter 1, the remainder of the Thesis is structured as follows. An overview of the relevant biological concepts is provided in Chapter 2.

Here, the current understanding of the structure of the plasma membrane, membrane proteins, and lipid monolayers are described. Most importantly, the complexity of biomembranes is highlighted in Chapter 2, and this complexity is connected to the peculiar observations on lipid and protein diﬀusion in Chapter 6.

The key theoretical concepts regarding lateral diﬀusion are described in Chapter 3.

Here, the theoretical models that are used to describe the diﬀusion of lipids and proteins are briefly introduced. Moreover, the concept of anomalous diﬀusion that is prevalent in biomembranes is discussed. A few theoretical descriptions that lead to anomalous dynamics are also presented. The relevance of lateral diﬀusion — including anomalous one — for cellular functions is justified. The main experimental methods that are commonly employed to tackle the questions related to lateral dynamics of membranes are also reviewed in Chapter 3. Their primary operating principles and limitations are discussed and their spatial and temporal resolutions are reviewed. Chapter 3 is closed by a justification for the need for computer simulations in the studies of membrane dynamics.

A brief look into the theoretical background of the molecular dynamics method employed throughout this Thesis is provided in Chapter 4. The fundamental concepts related to this methodology are introduced, and some of its central limitations are discussed. A thorough description of all the simulation models used in this Thesis is also provided at the end of Chapter 4.

Findings of this Thesis are described in Chapter 5 that is divided into three parts.

In the first one, the methodological contributions of this Thesis are described. This

5 work is covered by Publications I and II attached to this Thesis. In the second part of Chapter 5, the central results of this Thesis considering models describing normal diﬀusion of proteins and lipids are discussed. These findings have been reported in Publications III and IV attached to this Thesis. In the third section, the results of this Thesis on anomalous diﬀusion in membranes and monolayers are described.

This work includes both qualitative and quantitative analysis of the eﬀects of protein crowding and lipid packing on lipid and protein dynamics. This work is described in Publications V and VI attached to this Thesis.

A state-of-the-art picture of dynamics in biomembranes is provided in Chapter 6. The way the recent experimental and simulation eﬀorts have improved our understanding of the interplay between plasma membrane complexity and dynamics is systematically reviewed. To close this Thesis, some central open questions and possible future directions in the field are discussed.

6 1. Introduction

In document Diffusion of Lipids and Proteins in Complex Membranes (sivua 20-26)