From diversity of molecular forms to functional specialization of oligomeric proteins, nicotinic acetylcholine receptor, acetylcholinesterase and Na+, K+-ATPase

The review is devoted to the issue of diversity of molecular forms of oligomeric proteins using as examples members of the three protein classes: nicotinic acetylcholine receptor, acetylcholinesterase, and Na,K-ATPase. The data are presented on the molecular structure of proteins, subunit compositions, and isoforms of subunits, as well as on some features of gene expression. Particular emphasis has been made on the functional specialization of different molecular forms of one and the same oligomeric protein. The three above proteins, which serve seemingly quite different cellular processes, demonstrate many common principles of molecular mechanisms of physiological function.     

Driving membrane curvature in clathrin-dependent and clathrin-independent endocytosis

Cellular activity depends to a large extent on membrane bilayer dynamics. Many processes, such as organelle biogenesis and vesicular transport, rely on alterations in membrane structure and shape. It is now widely accepted that intracellular membrane curvature generation and remodelling is mediated and regulated by protein action, and the mechanisms behind the processes are currently being revealed. Here, we will briefly discuss the key principles of membrane deformation and focus on different endocytic events that use various kinds of proteins to shape the plasma membrane into transport carriers. The entry routes are adopted to make sure that a vast variety of molecules on the cell surface can be regulated by endocytosis. The principles for membrane sculpting of endocytic carriers can be viewed either from a perspective of rigid coat budding or of flexible opportunistic budding. We will discuss these principles and their implications, focusing on clathrin-dependent and -independent carrier formation and the proteins involved in the respective pathways.     

Using bleach-chase to measure protein half-lives in living cells

Protein removal has a central role in numerous cellular processes. Obtaining systematic measurements of multiple protein removal rates is necessary to understand the principles that govern these processes, but it is currently a major technical challenge. To address this, we developed 'bleach-chase', a noninvasive method for measuring the half-lives of multiple proteins at high temporal resolution in living cells. The method uses a library of annotated human reporter cell clones, each with a unique fluorescently tagged protein expressed from its native chromosomal location. In this protocol, we detail a simple procedure that bleaches the cells and uses time-lapse fluorescence microscopy and automated image analysis to systematically measure the half-life dynamics of multiple proteins. The duration of the protocol is 4-5 d. The method may be applicable to a wide range of fluorescently tagged proteins and cell lines.     

Formation of functional cell membrane domains: the interplay of lipid- and protein-mediated interactions

Numerous cell membrane associated processes, including signal transduction, membrane sorting, protein processing and virus trafficking take place in membrane subdomains. Protein-protein interactions provide the frameworks necessary to generate biologically functional membrane domains. For example, coat proteins define membrane areas destined for sorting processes, viral proteins self-assemble to generate a budding virus, and adapter molecules organize multimolecular signalling assemblies, which catalyse downstream reactions. The concept of raft lipid-based membrane domains provides a different principle for compartmentalization and segregation of membrane constituents. Accordingly, rafts are defined by the physical properties of the lipid bilayer and function by selective partitioning of membrane lipids and proteins into membrane domains of specific phase behaviour and lipid packing. Here, I will discuss the interplay of these independent principles of protein scaffolds and raft lipid microdomains leading to the generation of biologically functional membrane domains.     

Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins

Recent advances in generating active proteins through refolding of bacterial inclusion body proteins are summarized in conjunction with a short overview on inclusion body isolation and solubilization procedures. In particular, the pros and cons of well-established robust refolding techniques such as direct dilution as well as less common ones such as diafiltration or chromatographic processes including size exclusion chromatography, matrix- or affinity-based techniques and hydrophobic interaction chromatography are discussed. Moreover, the effect of physical variables (temperature and pressure) as well as the presence of buffer additives on the refolding process is elucidated. In particular, the impact of protein stabilizing or destabilizing low- and high-molecular weight additives as well as micellar and liposomal systems on protein refolding is illustrated. Also, techniques mimicking the principles encountered during in vivo folding such as processes based on natural and artificial chaperones and propeptide-assisted protein refolding are presented. Moreover, the special requirements for the generation of disulfide bonded proteins and the specific problems and solutions, which arise during process integration are discussed. Finally, the different strategies are examined regarding their applicability for large-scale production processes or high-throughput screening procedures.     

Protein quality control in the bacterial periplasm

The proper functioning of extracytoplasmic proteins requires their export to, and productive folding in, the correct cellular compartment. All proteins in Escherichia coli are initially synthesized in the cytoplasm, then follow a pathway that depends upon their ultimate cellular destination. Many proteins destined for the periplasm are synthesized as precursors carrying an N-terminal signal sequence that directs them to the general secretion machinery at the inner membrane. After translocation and signal sequence cleavage, the newly exported mature proteins are folded and assembled in the periplasm. Maintaining quality control over these processes depends on chaperones, folding catalysts, and proteases. This article summarizes the general principles which control protein folding in the bacterial periplasm by focusing on the periplasmic maltose-binding protein.     

Single-Molecule Force Spectroscopy of Membrane Protein Folding

Single-molecule force spectroscopy is a unique method that can probe the structural changes of single proteins at a high spatiotemporal resolution while mechanically manipulating them over a wide force range. Here, we review the current understanding of membrane protein folding learned by using the force spectroscopy approach. Membrane protein folding in lipid bilayers is one of the most complex biological processes in which diverse lipid molecules and chaperone proteins are intricately involved. The approach of single protein forced unfolding in lipid bilayers has produced important findings and insights into membrane protein folding. This review provides an overview of the forced unfolding approach, including recent achievements and technical advances. Progress in the methods can reveal more interesting cases of membrane protein folding and clarify general mechanisms and principles.     

The double-stranded RNA-binding motif, a versatile macromolecular docking platform

The double-stranded RNA-binding motif (dsRBM) is an alphabetabetabetaalpha fold with a well-characterized function to bind structured RNA molecules. This motif is widely distributed in eukaryotic proteins, as well as in proteins from bacteria and viruses. dsRBM-containing proteins are involved in processes ranging from RNA editing to protein phosphorylation in translational control and contain a variable number of dsRBM domains. The structural work of the past five years has identified a common mode of RNA target recognition by dsRBMs and dissected this recognition into two functionally separated interaction modes. The first involves the recognition of specific moieties of the RNA A-form helix by two protein loops, while the second is based on the interaction between structural elements flanking the RNA duplex with the first helix of the dsRBM. The latter interaction can be tuned by other protein elements. Recent work has made clear that dsRBMs can also recognize non-RNA targets (proteins and DNA), and act in combination with other dsRBMs and non-dsRBM motifs to play a regulatory role in catalytic processes. The elucidation of functional networks coordinated by dsRBM folds will require information on the precise functional relationship between different dsRBMs and a clarification of the principles underlying dsRBM-protein recognition.     

Chopped, trapped or tacked--protein translocation into the IMS of mitochondria

All proteins of the intermembrane space (IMS) of mitochondria are synthesized in the cytosol. The mechanisms by which these polypeptides are transported into the IMS are strikingly different from other protein-translocation processes in the cell. Recent studies suggest that IMS proteins reach their destination by three alternative principles that differ in the energy sources employed to drive the translocation reactions. The first class of proteins uses both hydrolysis of matrix ATP and the electrochemical potential of the inner membrane. The second class depends on the energy gain of protein folding, and the third on the association of the proteins to high-affinity binding sites in the IMS.     

Trafficking of STEVOR to the Maurer's clefts in Plasmodium falciparum-infected erythrocytes

The human malarial parasite Plasmodium falciparum exports proteins to destinations within its host erythrocyte, including cytosol, surface and membranous profiles of parasite origin termed Maurer's clefts. Although several of these exported proteins are determinants of pathology and virulence, the mechanisms and trafficking signals underpinning protein export are largely uncharacterized-particularly for exported transmembrane proteins. Here, we have investigated the signals mediating trafficking of STEVOR, a family of transmembrane proteins located at the Maurer's clefts and believed to play a role in antigenic variation. Our data show that, apart from a signal sequence, a minimum of two addition signals are required. This includes a host cell targeting signal for export to the host erythrocyte and a transmembrane domain for final sorting to Maurer's clefts. Biochemical studies indicate that STEVOR traverses the secretory pathway as an integral membrane protein. Our data suggest general principles for transport of transmembrane proteins to the Maurer's clefts and provide new insights into protein sorting and trafficking processes in P. falciparum.     

Function and regulation of protein neddylation. 'Protein modifications: beyond the usual suspects' review series

Neddylation is the post-translational protein modification that is most closely related to ubiquitination. However, ubiquitination is known to regulate a myriad of processes in eukaryotic cells, whereas only a limited number of neddylation substrates have been described to date. Here, we review the principles of protein neddylation and highlight the mechanisms that ensure the specificity of neddylation over ubiquitination. As numerous neddylation substrates probably remain to be discovered, we propose some criteria that could be used as guidelines for the characterization of neddylated proteins.     

酵母双杂交系统在膜蛋白相互作用研究中的应用

膜相关蛋白约占细胞总蛋白质中的1/3,它们大都参与了细胞的诸多生理、病理过程和药物反应机理。研究膜蛋白的相互作用对于揭示细胞的生命活动规律及寻找药物作用靶标都有重要的意义。由于膜蛋白本身的特性及其难以进入核内等原因,经典的酵母双杂交技术并不适用于检测膜蛋白间的相互作用。针对在活细胞中研究膜蛋白相互作用的需要,近年来国际上先后发展了一系列用于膜蛋白相互作用研究的酵母双杂交新系统,并取得了许多重要发现。     

The analysis of protein structures: New insights from a growing data base

We now know the structures of over 200 proteins to atomic resolution. Despite the impressive extent and quality of the results, crystal-structure analysis has often been thought of as limited in scope, not only in its restriction to samples that can be crystallized, but in the more important respect that taking ‘snapshots’ of proteins does not directly address the complex spatio-temporal organization of the processes in which proteins participate. It is suggested here that, as the field has matured, this second limitation is gradually being overcome. As we gain increased access to structures of proteins in different conformational states – for example, in conformations produced by different states of ligation – and to families of homologous proteins, we can proceed from the statics of protein structure to the dynamics of conformational change, function, and evolution. A new scientific speciality has grown up around the solved structures: it has as its goal the elucidation of general principles of protein structure and function, to provide a theoretical framework for understanding the properties of proteins revealed by experiment. In this article we shall discuss some of the activity in this field. It will emerge clearly, I believe, that the increasing number and variety of solved structures is exerting a cumulative force. General principles are emerging from comparisons of related proteins and contrasts of dissimilar ones: the whole corpus of data is greater than the sum of the parts.     

Membrane interactions of intrinsically disordered proteins: The example of alpha-synuclein

Peripheral membrane proteins associate reversibly with biological membranes that, compared to protein binding partners, are structurally labile and devoid of specific binding pockets. Membranes in different subcellular compartments vary primarily in their chemical composition and physical properties, and recognition of these features is therefore critical for allowing such proteins to engage their proper membrane targets. Intrinsically disordered proteins (IDPs) are well-suited to accomplish this task using highly specific and low- to moderate-affinity interactions governed by recognition principles that are both similar to and different from those that mediate the membrane interactions of rigid proteins. IDPs have also evolved multiple mechanisms to regulate membrane (and other) interactions and achieve their impressive functional diversity. Moreover, IDP-membrane interactions may have a kinetic advantage in fast processes requiring rapid control of such interactions, such as synaptic transmission or signaling. Herein we review the biophysics, regulation and functional implications of IDP-membrane interactions and include a brief overview of some of the methods that can be used to study such interactions. At each step, we use the example of alpha-synuclein, a protein involved in the pathogenesis of Parkinson’s disease and one of the best characterized membrane-binding IDP, to illustrate some of the principles discussed.     

Membrane fusion

Membrane fusion, one of the most fundamental processes in life, occurs when two separate lipid membranes merge into a single continuous bilayer. Fusion reactions share common features, but are catalyzed by diverse proteins. These proteins mediate the initial recognition of the membranes that are destined for fusion and pull the membranes close together to destabilize the lipid/water interface and to initiate mixing of the lipids. A single fusion protein may do everything or assemblies of protein complexes may be required for intracellular fusion reactions to guarantee rigorous regulation in space and time. Cellular fusion machines are adapted to fit the needs of different reactions but operate by similar principles in order to achieve merging of the bilayers.     

The membrane-water interface region of membrane proteins: structural bias and the anti-snorkeling effect

Membrane proteins have important roles in many cellular processes. Computational analysis of their sequences and structures has provided much insight into the organizing principles of transmembrane helices. In a recent study, the membrane-water interface region was examined in detail for the first time. The results have revealed that this interface region has an important role in constraining protein secondary structure. This study raises new questions and opens up new directions for studying membrane proteins.