Tag–probe labeling methods for live-cell imaging of membrane proteins

Instead of using reconstituted proteoliposomes, in situ investigations of membrane proteins in living cell membranes are important because the heterogeneous and dynamic nature of biomembranes significantly affects their behavior. Protein-specific labeling is a key technique for the detection of a target protein by fluorescence measurements, particularly fluorescence microscopy. However, conventional genetic fusion with fluorescent proteins has several shortcomings. Post-translational labeling methods using a genetically encodable tag and synthetic probes targeting to the tag can overcome these limitations. This review summarizes emerging tag–probe techniques for labeling specific membrane proteins and their applications, including endocytotic internalization, partitioning to specific membrane domains, interprotein interactions, and conformational changes.     

Trafficking of phosphatidylinositol by phosphatidylinositol transfer proteins

PtdIns is synthesized at the endoplasmic reticulum and its intracellular distribution to other organelles can be facilitated by lipid transfer proteins [PITPs (phosphatidylinositol transfer proteins)]. In this review, I summarize the current understanding of how PITPs are regulated by phosphorylation, how can they dock to membranes to exchange their lipid cargo and how cells use PITPs in signal transduction and membrane delivery. Mammalian PITPs, PITPalpha and PITPbeta, are paralogous genes that are 94% similar in sequence. Their structural design demonstrates that they can sequester PtdIns or PtdCho (phosphatidylcholine) in their hydrophobic cavity. To deliver the lipid cargo to a membrane, PITP has to undergo a conformational change at the membrane interface. PITPs have a higher affinity for PtdIns than PtdCho, which is explained by hydrogen-bond contacts between the inositol ring of PtdIns and the side-chains of four amino acid residues, Thr59, Lys61, Glu86 and Asn90, in PITPs. Regardless of species, these residues are conserved in all known PITPs. PITP transfer activity is regulated by a conserved serine residue (Ser166) that is phosphorylated by protein kinase C. Ser166 is only accessible for phosphorylation when a conformational change occurs in PITPs while docking at the membrane interface during lipid transfer, thereby coupling regulation of activity with lipid transfer function. Biological roles of PITPs include their ability to couple phospholipase C signalling to neurite outgrowth, cell division and stem cell growth.     

Quantitative understanding of the energy transfer between fluorescent proteins connected via flexible peptide linkers

The fusion of different protein domains via peptide linkers is a powerful, modular approach to obtain proteins with new functions. A detailed understanding of the conformational behavior of peptide linkers is important for applications such as fluorescence resonance energy transfer (FRET)-based sensor proteins and multidomain proteins involved in multivalent interactions. To investigate the conformational behavior of flexible glycine- and serine-containing peptide linkers, we constructed a series of fusion proteins of enhanced cyan and yellow fluorescent proteins (ECFP-linker-EYFP) in which the linker length was systematically varied by incorporating between 1 and 9 GGSGGS repeats. As expected, both steady-state and time-resolved fluorescence measurements showed a decrease in energy transfer with increasing linker length. The amount of energy transfer observed in these fusion proteins can be quantitatively understood by simple models that describe the flexible linker as a worm-like chain with a persistence length of 4.5 A or a Gaussian chain with a characteristic ratio of 2.3. The implications of our results for understanding the properties of FRET-based sensors and other fusion proteins with Gly/Ser linkers are discussed.     

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels², ion pumps³, and transporters⁴. Recent developments have combined site-specific fluorophore labeling alongside TEVC to concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling^5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues^7,8. This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal⁵. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins⁴. Finally, recent developments have also enabled the manipulation of select internal ions following co-expression with a second protein⁹.Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (<5%)³ upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of the membrane protein in order to correlate the conformational dynamics to the function of the protein¹⁰. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.     

Strategies for membrane interaction proteomics: no mass spectrometry required

Membrane-bound proteins are one of the most important protein types in the cell, and are involved in many major cell processes and signaling pathways. Most proteins, including those at membranes, must interact with other proteins to form complexes, which are essential for their function(s). In this review, we describe some of the major non-mass spectrometry-based methods and technologies used for the investigation of intracellular membrane protein complexes including Tango, fluorescence/bioluminescence resonance energy transfer (F/BRET), luminescence-based mammalian interactome mapping (LUMIER), protein-fragment complementation assay (PCA), and membrane yeast two-hybrid assay (MYTH). We highlight the advantages and drawbacks of these methods, describe recent studies utilizing these methods, and discuss some of the major findings in the study of membrane protein-based cell pathways.     

Conformational dynamics of two histidine-binding proteins of Salmonella typhimurium.

The Salmonella typhimurium periplasmic histidine-binding J-protein is one of four proteins encoded by the histidine transport operon. Mutant J-protein hisJ5625 binds L-histidine, but does not transport it. The tertiary structure and conformational dynamics of native and mutant J-protein have been compared using steady state fluorescence, fluorescence polarization, and fluorescence energy transfer measurements. The two proteins have different three-dimensional structures and exhibit different responses to histidine binding. Ligand-induced conformational changes were demonstrated in both J-proteins using fluorescence energy transfer (distant reporter method) between the single tryptophan residue per mole of protein and a fluorescein-labeled methionine residue. However, the conformational change of the mutant protein is qualitatively and quantitatively different from that of the wild-type protein. Moreover, the microenvironment of the tryptophan and its distance from the labeled methionine (44A for the wild type, 60A for the mutant J-protein) are different in the two proteins. In conclusion, these results indicate that the specific conformational change induced in the wild type J-protein is a necessary requirement for the transport of L-histidine.     

Imaging molecular interactions in living cells

Hormones integrate the activities of their target cells through receptor-modulated cascades of protein interactions that ultimately lead to changes in cellular function. Understanding how the cell assembles these signaling protein complexes is critically important to unraveling disease processes, and to the design of therapeutic strategies. Recent advances in live-cell imaging technologies, combined with the use of genetically encoded fluorescent proteins, now allow the assembly of these signaling protein complexes to be tracked within the organized microenvironment of the living cell. Here, we review some of the recent developments in the application of imaging techniques to measure the dynamic behavior, colocalization, and spatial relationships between proteins in living cells. Where possible, we discuss the application of these different approaches in the context of hormone regulation of nuclear receptor localization, mobility, and interactions in different subcellular compartments. We discuss measurements that define the spatial relationships and dynamics between proteins in living cells including fluorescence colocalization, fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy. These live-cell imaging tools provide an important complement to biochemical and structural biology studies, extending the analysis of protein-protein interactions, protein conformational changes, and the behavior of signaling molecules to their natural environment within the intact cell.     

Conformational changes in protein loops and helices induced by post-translational phosphorylation

Post-translational phosphorylation is a ubiquitous mechanism for modulating protein activity and protein-protein interactions. In this work, we examine how phosphorylation can modulate the conformation of a protein by changing the energy landscape. We present a molecular mechanics method in which we phosphorylate proteins in silico and then predict how the conformation of the protein will change in response to phosphorylation. We apply this method to a test set comprised of proteins with both phosphorylated and non-phosphorylated crystal structures, and demonstrate that it is possible to predict localized phosphorylation-induced conformational changes, or the absence of conformational changes, with near-atomic accuracy in most cases. Examples of proteins used for testing our methods include kinases and prokaryotic response regulators. Through a detailed case study of cyclin-dependent kinase 2, we also illustrate how the computational methods can be used to provide new understanding of how phosphorylation drives conformational change, why substituting Glu or Asp for a phosphorylated amino acid does not always mimic the effects of phosphorylation, and how a phosphatase can “capture” a phosphorylated amino acid. This work illustrates how computational methods can be used to elucidate principles and mechanisms of post-translational phosphorylation, which can ultimately help to bridge the gap between the number of known sites of phosphorylation and the number of structures of phosphorylated proteins.     

Visualization of ternary complexes in living cells by using a BiFC-based FRET assay

Studies of protein interactions have increased our understanding and knowledge of biological processes. Assays that utilize fluorescent proteins, such as fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC), have enabled direct visualization of protein interactions in living cells. However, these assays are primarily suitable for a pair of interacting proteins, and methods to visualize and identify multiple protein complexes in vivo are very limited. This protocol describes the recently developed BiFC-FRET assay, which allows visualization of ternary complexes in living cells. We discuss how to design the BiFC-FRET assay on the basis of the validation of BiFC and FRET assays and how to perform transfection experiments for acquisition of fluorescent images for net FRET calculation. We also provide three methods for normalization of the FRET efficiency. The assay employs a two-chromophore and three-filter FRET setup and is applicable to epifluorescence microscopes. The entire protocol takes about 2-3 weeks to complete.     

Diving Into the Lipid Bilayer to Investigate the Transmembrane Organization and Conformational State Transitions of P-type Ion ATPases

Although membrane proteins constitute more than 20% of the total proteins, the structures of only a few are known in detail. An important group of integral membrane proteins are ion-transporting ATPases of the P-type family, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. There are several crystal structures of the sarcoplasmic reticulum Ca(2+) pump (SERCA) revealing different conformations, and recently, crystal structures of the H(+)-ATPase and the Na(+)/K(+)-ATPase were reported as well. However, there are no atomic resolution structures for other P-type ATPases including the plasma membrane calcium pump (PMCA), which is integral to cellular Ca(2+) signaling. Crystallization of these proteins is challenging because there is often no natural source from which the protein can be obtained in large quantities, and the presence of multiple isoforms in the same tissue further complicates efforts to obtain homogeneous samples suitable for crystallization. Alternative techniques to study structural aspects and conformational transitions in the PMCAs (and other P-type ATPases) have therefore been developed. Specifically, information about the structure and assembly of the transmembrane domain of an integral membrane protein can be obtained from an analysis of the lipid-protein interactions. Here, we review recent efforts using different hydrophobic photo-labeling methods to study the non-covalent interactions between the PMCA and surrounding phospholipids under different experimental conditions, and discuss how the use of these lipid probes can reveal valuable information on the membrane organization and conformational state transitions in the PMCA, Na(+)/K(+)-ATPase, and other P-type ATPases.     

A computational tool for designing FRET protein biosensors by rigid-body sampling of their conformational space

Biosensors relying on the fluorescence resonance energy transfer (FRET) between fluorescent proteins have been used for live-cell imaging of cellular events including Ca(2+) signaling. The efficiency of energy transfer between the donor and acceptor fluorescent proteins depends on the relative distance and orientation between them, which become altered by conformational changes of a fused sensory protein caused by a cellular event. In this way, changes in FRET efficiency of Ca(2+) biosensors can be correlated with Ca(2+) concentrations. The design of these FRET biosensors can be improved by modeling conformational changes before and after a cellular event. Hence, a computational tool called FPMOD was developed to predict FRET efficiency changes by constructing FRET biosensors and sampling their conformational space through rigid-body rotation. We showed with FPMOD that our computational modeling approach can qualitatively predict the FRET efficiencies of a range of biosensors, which had strong agreement with experimental results.     

A functional NMR for membrane proteins: dynamics,ligand binding,and allosteric modulation

By nature of conducting ions, transporting substrates and transducing signals, membrane channels, transporters and receptors are expected to exhibit intrinsic conformational dynamics. It is therefore of great interest and importance to understand the various properties of conformational dynamics acquired by these proteins, for example, the relative population of states, exchange rate, conformations of multiple states, and how small molecule ligands modulate the conformational exchange. Because small molecule binding to membrane proteins can be weak and/or dynamic, structural characterization of these effects is very challenging. This review describes several NMR studies of membrane protein dynamics, ligand‐induced conformational rearrangements, and the effect of ligand binding on the equilibrium of conformational exchange. The functional significance of the observed phenomena is discussed.     

Calmodulin, conformational states, and calcium signaling. A single-molecule perspective

Single-molecule fluorescence measurements can provide a new perspective on the conformations, dynamics, and interactions of proteins. Recent examples are described illustrating the application of single-molecule fluorescence spectroscopy to calcium signaling proteins with an emphasis on the new information available in single-molecule fluorescence burst measurements, resonance energy transfer, and polarization modulation methods. Calcium signaling pathways are crucial in many cellular processes. The calcium binding protein calmodulin (CaM) serves as a molecular switch to regulate a network of calcium signaling pathways. Single-molecule spectroscopic methods can yield insights into conformations and dynamics of CaM and CaM-regulated proteins. Examples include studies of the conformations and dynamics of CaM, binding of target peptides, and interaction with the plasma-membrane Ca2+ pump. Single-molecule resonance energy transfer measurements revealed conformational substates of CaM, and single-molecule polarization modulation spectroscopy was used to probe interactions between CaM and the plasma-membrane Ca2+-ATPase.     

Physicochemical bases for protein folding,dynamics, and protein-ligand binding

Proteins are essential parts of living organisms and participate in virtually every process within cells.As the genomic sequences for increasing number of organisms are completed,research into how proteins can perform such a variety of functions has become much more intensive because the value of the genomic sequences relies on the accuracy of understanding the encoded gene products.Although the static three-dimensional structures of many proteins are known,the functions of proteins are ultimately governed by their dynamic characteristics,including the folding process,conformational fluctuations,molecular motions,and protein-ligand interactions.In this review,the physicochemical principles underlying these dynamic processes are discussed in depth based on the free energy landscape(FEL)theory.Questions of why and how proteins fold into their native conformational states,why proteins are inherently dynamic,and how their dynamic personalities govern protein functions are answered.This paper will contribute to the understanding of structure-function relationship of proteins in the post-genome era of life science research.     

A conformational change in the alpha-subunit of coatomer induced by ligand binding to gamma-COP revealed by single-pair FRET

Formation of transport vesicles involves polymerization of cytoplasmic coat proteins (COP). In COPI vesicle biogenesis, the heptameric complex coatomer is recruited to donor membranes by the interaction of multiple coatomer subunits with the budding machinery. Specific binding to the trunk domain of γ-COP by the Golgi membrane protein p23 induces a conformational change that causes polymerization of the complex. Using single-pair fluorescence resonance energy transfer, we find that this conformational change takes place in individual coatomer complexes, independent of each other, and that the conformational rearrangement induced in γ-COP is transmitted within the complex to its α-subunit. We suggest that capture of membrane protein machinery triggers cage formation in the COPI system.     

Investigating the mechanisms of photosynthetic proteins using continuum electrostatics

Computational methods based on continuum electrostatics are widely used in theoretical biochemistry to analyze the function of proteins. Continuum electrostatic methods in combination with quantum chemical and molecular mechanical methods can help to analyze even very complex biochemical systems. In this article, applications of these methods to proteins involved in photosynthesis are reviewed. After giving a short introduction to the basic concepts of the continuum electrostatic model based on the Poisson-Boltzmann equation, we describe the application of this approach to the docking of electron transfer proteins, to the comparison of isofunctional proteins, to the tuning of absorption spectra, to the analysis of the coupling of electron and proton transfer, to the analysis of the effect of membrane potentials on the energetics of membrane proteins, and to the kinetics of charge transfer reactions. Simulations as those reviewed in this article help to analyze molecular mechanisms on the basis of the structure of the protein, guide new experiments, and provide a better and deeper understanding of protein functions.     

Fluorescence characterization of denatured proteins

Characterization of unfolded states, while critical to a complete understanding of protein folding, is inherently difficult due to structural heterogeneity and dynamic interchange between states. The growing body of work focusing on single molecule fluorescence techniques for the study of protein folding, also highlights their potential for studies of unfolded proteins. These methods can obtain conformational information about individual subpopulations of molecules in an ensemble, and measure dynamics without the need for synchronization. The studies highlighted here demonstrate the promise of these techniques for obtaining novel information about unfolded states in vitro and in more physiologically relevant milieu.     

Fluorescence-based methods to image palmitoylated proteins

A well known function of palmitoylation is to promote protein binding to cell membranes. Until recently, it was unclear what additional roles, if any, palmitoylation has in controlling protein localization in cells. Recent studies of palmitoylated forms of the small GTPase Ras have now revealed that palmitoylation plays multiple roles in the regulation of protein trafficking, including targeting proteins into the secretory pathway and recycling proteins between the plasma membrane and Golgi complex. We here describe how quantitative fluorescence microscopy and photobleaching approaches can be used to study the intracellular targeting and trafficking of GFP-tagged palmitoylated proteins in living cells. We discuss (1) general considerations for fluorescence recovery after photobleaching (FRAP) measurements of GFP-tagged proteins; (2) FRAP-based assays to test the strength of binding of palmitoylated proteins to cell membranes; (3) methods to establish the kinetics and mechanisms of recycling of palmitoylated proteins between the Golgi complex and the plasma membrane; (4) the use of the palmitoylation inhibitor 2-bromo-palmitate as a tool to study the dynamic regulation of protein targeting and trafficking by palmitate turnover.     

Photobleaching approaches to investigate diffusional mobility and trafficking of Ras in living cells

Recent advances in our understanding of the intracellular trafficking, membrane microenvironment, and subcellular sites of signaling of Ras have been driven by observations of GFP-tagged Ras in living cells. Here, we describe methods to gain further insight into the regulation of these events through the use of quantitative fluorescence microscopy. We focus on three techniques, fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), and selective photobleaching. While all of these techniques exploit photobleaching as a tool to monitor protein dynamics, they each provide a unique subset of information. In particular, FRAP provides measurements of protein mobility via lateral diffusion by monitoring recovery of fluorescence into a region following a single photobleaching event. FLIP assesses the level of continuity and communication between subcellular compartments by repetitively photobleaching a region of interest and following concomitant loss of fluorescence from other areas in the cell. Selective photobleaching reveals kinetic information about active and passive transport of proteins into organelles such as the Golgi complex or between areas of protein enrichment such as caveolae. We describe how to implement these techniques using commercially available confocal microscopes and outline methods for data analysis. Finally, we discuss how these approaches are being used to provide new insights into the mechanisms of membrane microdomain localization, vesicular versus non-vesicular transport, and kinetics of exchange of Ras on and off of cell membranes.     

New approaches towards the understanding of integral membrane proteins: A structural perspective on G protein‐coupled receptors

Three‐dimensional structure determination of integral membrane proteins has advanced in unprecedented detail our understanding of mechanistic events of how ion channels, transporters, receptors, and enzymes function. This exciting progress required a tremendous amount of methods development, as exemplified here with G protein‐coupled receptors (GPCRs): Optimizing the production of GPCRs in recombinant hosts; increasing the probability of crystal formation using high‐affinity ligands, nanobodies, and minimal G proteins for co‐crystallization, thus stabilizing receptors into one conformation; using the T4 lysozyme technology and other fusion partners to promote crystal contacts; advancing crystallization methods including the development of novel detergents, and miniaturization and automation of the lipidic cubic phase crystallization method; the concept of conformational thermostabilization of GPCRs; and developing microfocus X‐ray synchrotron technologies to analyze small GPCR crystals. However, despite immense progress to explain how GPCRs function, many receptors pose intractable hurdles to structure determination at this time. Three emerging methods, serial femtosecond crystallography, micro electron diffraction, and single particle electron cryo‐microscopy, hold promise to overcome current limitations in structural membrane biology.