Current Protein and Peptide Science - Volume 12, Issue 8, 2011

One third of the genome of any organism encodes intrinsic membrane proteins. Along with peripheral membrane proteins and proteins that only transiently interact with the membrane, these molecules are hard to study but of great importance to understand both the normal and pathological life of the cell. Biophysical methods are of the utmost importance when trying to unravel the molecular details of processes that have been described by biochemical methods. In particular, biophysical methods are particularly well suited to study membrane proteins. We present here a sample of current understanding of membrane-associated proteins function and structure, studied by state-of-the- art biophysical, biochemical and modeling techniques. In the manuscript by Borioli, a novel hypothesis about immediateearly proto-oncoproteins structure-function relationship in regard to their ability to transiently interact with membranes is discussed. Colombo and Fasana discuss recent data regarding the unassisted insertion of tail-anchored proteins into the membrane of the endoplasmic reticulum. Cybulski and de Mendoza's manuscript stresses the importance of bilayer thickness for membrane protein function. We present four manuscripts that use single-molecule methods to understand membrane-protein, protein-protein and carbohydrate- protein interactions in membranes, highlighting the increasing importance of these techniques for the development of the field. The manuscript by Bleicken and co-workers describes the use of Fluorescence Correlation Spectroscopy to address the experimentally challenging study of the interaction between membrane proteins as they find each other in the bilayer. Clausen and Lagerholm discuss in detail the effect of the probe size and photophysics in the results obtained from single particle tracking of probes attached to membrane components. Ruprecht and co-workers present a new way to analyze single molecule diffusion in the absence of a full analytical treatment. The method is based on the comparison of an experimental data set against the outcome of multiple experiments performed on the computer by Monte Carlo simulations. The manuscript by Leckband emphasizes the unique information obtained by force measurements on the binding mechanisms of lectins to cell-surface carbohydrates. Finally, Banning and co-authors summarize the recent findings on Reggie/Flotillin mechanisms of association in membrane microdomains and in oligomers. I am excited to deliver this issue that tackles a diverse set of developments in the area of the biophysics of membraneassociated proteins. I hope that it will be interesting for biophysicists, cell biologists and biochemists. I would like to thank all the authors who made this issue possible. I am in great debt to the anonymous reviewers from around the world who delivered timely and useful comments to the authors. I used here a double-blind method which I hope contributed to make the reviewing process more objective than it usually is. I also express my gratitude to the Editor-in-Chief Prof. Ben M. Dunn for his invitation and kind support that resulted in the successful completion of this special issue. Last but not least, a message of gratitude to the Assistant Manager of Bentham Science Publishers Sadia Rafique, who helped me with great efficiency to put together all the last details of the issue.

Proto-oncoproteins are a heterogeneous group of proteins that induce cellular differentiation, proliferation and growth, acting at different points of signaling cascades and in different cell compartments, through many different mechanisms. If the proto-oncogenes that give raise to proto-oncoproteins undergo genetic damage, they become oncogenes and their products are the oncoproteins responsible for cellular transformation in cancer. Some proto-oncoproteins are related to membranes and they exert their function at this level. Among these are receptors and receptor-like growth factors, membrane associated tyrosine kinases, and small GTPases. Other proto-oncoproteins are transcription factors and as such, their best known functional context is promoter DNA regions. Consequently, DNA is widely viewed as their most relevant non protein partner. Any interaction of these proteins with membranes is generally overlooked and, when considered, the membrane is regarded as a reservoir for timely release requiring proteolytic activity. However, this status quo should be revised. Some Immediate-Early proteins that are mobilized in the cell shortly after stimulus are also a subset of the transcription factor kind of proto-oncoproteins. These particular Immediate-Early proto- Oncoproteins (IEOs) exceed strict DNA related functions. Gathering evidence coming from biophysical studies on one hand and from molecular and cellular studies on the other hand, converge suggesting a link between them and membranes. In this review we discuss the conception that transcription factors with the features of IEOs exert their function in cellular processes, not only through association to DNA related to targeted transcription, but also through association to membranes related to global replication and transcription.

The characterization of interactions between membrane proteins as they take place within the lipid bilayer poses a technical challenge, which is currently very difficult and, in many cases, impossible to overcome. The recent development of a method based in the combination two-color fluorescence cross-correlation spectroscopy with scanning of the focal volume allows the detection and quantification of interactions between biomolecules inserted in biological membranes. This powerful strategy has allowed the quantitative analysis of diverse systems, such as the association between proteins of the Bcl-2 family involved in apoptosis regulation or the binding between a growth factor and its receptor during signaling. Here, we review the last developments to quantify protein/protein interactions in lipid membranes and focus on the use of fluorescence-correlation-spectroscopy approaches for that purpose.

Single particle tracking (SPT) enables light microscopy at a sub-diffraction limited spatial resolution by a combination of imaging at low molecular labeling densities and computational image processing. SPT and related single molecule imaging techniques have found a rapidly expanded use within the life sciences. This expanded use is due to an increased demand and requisite for developing a comprehensive understanding of the spatial dynamics of bio-molecular interactions at a spatial scale that is equivalent to the size of the molecules themselves, as well as by the emergence of new imaging techniques and probes that have made historically very demanding and specialized bio-imaging techniques more easily accessible and achievable. SPT has in particular found extensive use for analyzing the molecular organization of biological membranes. From these and other studies using complementary techniques it has been determined that the organization of native plasma membranes is heterogeneous over a very large range of spatial and temporal scales. The observed heterogeneities in the organization have the practical consequence that the SPT results in investigations of native plasma membranes are time dependent. Furthermore, because the accessible time dynamics, and also the spatial resolution, in an SPT experiment is mainly dependent on the luminous brightness and photostability of the particular SPT probe that is used, available SPT results are ultimately dependent on the SPT probes. The focus of this review is on the impact that the SPT probe has on the experimental results in SPT.

Diffusing membrane constituents are constantly exposed to a variety of forces that influence their stochastic path. Single molecule experiments allow for resolving trajectories at extremely high spatial and temporal accuracy, thereby offering insights into en route interactions of the tracer. In this review we discuss approaches to derive information about the underlying processes, based on single molecule tracking experiments. In particular, we focus on a new versatile way to analyze single molecule diffusion in the absence of a full analytical treatment. The method is based on comprehensive comparison of an experimental data set against the hypothetical outcome of multiple experiments performed on the computer. Since Monte Carlo simulations can be easily and rapidly performed even on state-of-the-art PCs, our method provides a simple way for testing various - even complicated - diffusion models. We describe the new method in detail, and show the applicability on two specific examples: firstly, kinetic rate constants can be derived for the transient interaction of mobile membrane proteins; secondly, residence time and corral size can be extracted for confined diffusion.

Flotillin-2 and flotillin-1, also called reggie-1 and reggie-2, are ubiquitously expressed and highly conserved proteins. Originally, they were described as neuronal regeneration proteins, but they appear to function in a wide variety of cellular processes, such as membrane receptor signaling, endocytosis, phagocytosis and cell adhesion. The molecular details of the function of flotillins in these processes have only been partially clarified. Flotillins are associated with cholesterol and sphingolipid enriched membrane microdomains known as rafts, and some findings even suggest that they define their own kind of a microdomain. The mechanism of the membrane association of flotillins appears to rely mainly on acylation (myristoylation and/or palmitoylation), localizing flotillins onto the cytosolic side of the membranes, whereas no transmembrane domains are present. In addition, flotillins show a strong tendency to form homo- and hetero-oligomers with each other. In this review, we will summarize the recent findings on the function of flotillins and discuss the mechanisms that might regulate their function, such as membrane association, oligomerization and phosphorylation.

Tail-anchored proteins (TAPs) are a subclass of type II integral membrane proteins that carry out important and diverse functions within cells. Structurally, TAPs present an N-terminal domain exposed to the cytosol and a single transmembrane domain (TMD) close to the C-terminus, the latter is responsible for the targeting and insertion into the proper intracellular membrane (endoplasmic reticulum (ER), mitochondria, peroxisomes). Due to this particular topology, TAPs insert obligatorily into membranes by post-translational pathways and are excluded from the classical SRP dependent co-translational ER insertion. ER-targeted TAPs can follow two distinct ways of insertion according to the hydrophobicity of their TMD. In the “assisted” pathway, TAPs with more hydrophobic TMDs insert in the ER membrane with the requirement of energy and the involvement of proteinaceous component(s). By contrast neither energy, nor membrane or cytosolic proteins are necessary and do not even improve the “unassisted” insertion of TAPs with moderately hydrophobic TMDs. In this review, we discuss the most relevant recent data regarding the molecular mechanism that underlies these processes.

Cell surface carbohydrates are important targets for many cell surface receptors, and they mediate crucial biological processes ranging from pathogen infectivity to neutrophil adhesion to drug targeting. A central challenge is to identify relationships between lectin architecture and function that influence the adhesion strength, avidity, and kinetics of receptor-glycan bonds. This information is central both to understanding recognition mechanisms and to developing effective therapeutic agents for drug targeting or for preventing infection. Increasingly, force probes are used to assess structure activity relationships of both the glycan ligands and the receptors that bind them, as well as molecular mechanisms underlying binding and adhesion. This review describes recent advances in the use of different force measurement techniques to quantify receptor-glycan bond parameters, and to identify novel features of molecular mechanisms underlying recognition and adhesion. The examples discussed focus in particular on single bond rupture, surface force measurements, and micropipette manipulation. This review emphasizes the often-unique information obtained from studies of lectin interactions with carbohydrate ligands that complement more common structure determinations and solution binding studies.

The influence of the lipid environment on the function of membrane proteins is increasingly recognized as crucial. Nevertheless, the molecular mechanisms underlying protein-lipid interactions remain obscure. Membrane lipid composition has a regulatory effect on membrane protein activity, and for a number of membrane proteins a clear correlation was found between protein activity and properties of the membrane bilayer such as fluidity. Membrane thickness is an important property of a lipid bilayer. It is expected that hydrophobic thickness match the hydrophobic thickness of transmembrane segments of integral membrane proteins. Any mismatch between the hydrophobic thicknesses of the lipid bilayer and the protein would lead to some modification in either the structure of the protein or the structure of the bilayer, or both. Consequent rearrangements may result in changes in protein activity. Here we review the behavior of several transmembrane proteins whose activity is altered by hydrophobic core thickness.