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Methods in Molecular Biology (Clifton,... 2021Protein engineering can yield new molecular tools for nanotechnology and therapeutic applications through modulating physiochemical and biological properties....
Protein engineering can yield new molecular tools for nanotechnology and therapeutic applications through modulating physiochemical and biological properties. Engineering membrane proteins is especially attractive because they perform key cellular processes including transport, nutrient uptake, removal of toxins, respiration, motility, and signaling. In this chapter, we describe two protocols for membrane protein engineering with the Rosetta software: (1) ΔΔG calculations for single point mutations and (2) sequence optimization in different membrane lipid compositions. These modular protocols are easily adaptable for more complex problems and serve as a foundation for efficient membrane protein engineering calculations.
Topics: Biological Transport; Membrane Lipids; Membrane Proteins; Membranes; Models, Molecular; Protein Engineering; Software
PubMed: 34302669
DOI: 10.1007/978-1-0716-1468-6_3 -
Current Opinion in Structural Biology Oct 2019Solving high-resolution structures of membrane proteins has been an important challenge for decades, still lagging far behind that of soluble proteins even with the... (Review)
Review
Solving high-resolution structures of membrane proteins has been an important challenge for decades, still lagging far behind that of soluble proteins even with the recent remarkable technological advances in X-ray crystallography and electron microscopy. Central to this challenge is the necessity to isolate and solubilize membrane proteins in a stable, natively folded and functional state, a process influenced by not only the proteins but also their surrounding chemical environment. This review highlights recent community efforts in the development and characterization of novel membrane agents and ligand tools to stabilize individual proteins and protein complexes, which together have accelerated progress in membrane protein structural biology.
Topics: Crystallization; Detergents; Membrane Proteins; Protein Stability; Solubility
PubMed: 31285102
DOI: 10.1016/j.sbi.2019.06.002 -
Biochimica Et Biophysica Acta.... Jan 2020Membrane protein folding studies lag behind those of water-soluble proteins due to immense difficulties of experimental study, resulting from the need to provide a... (Review)
Review
Membrane protein folding studies lag behind those of water-soluble proteins due to immense difficulties of experimental study, resulting from the need to provide a hydrophobic lipid-bilayer environment when investigated in vitro. A sound understanding of folding mechanisms is important for membrane proteins as they contribute to a third of the proteome and are frequently associated with disease when mutated and/or misfolded. Membrane proteins largely consist of α-helical, hydrophobic transmembrane domains, which insert into the membrane, often using the SecYEG/Sec61 translocase system. This mini-review highlights recent advances in techniques that can further our understanding of co-translational folding and notably, the structure and insertion of nascent chains as they emerge from translating ribosomes. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.
Topics: Animals; Humans; Membrane Proteins; Protein Folding; Protein Translocation Systems; Ribosomes; SEC Translocation Channels
PubMed: 31302079
DOI: 10.1016/j.bbamem.2019.07.007 -
BMC Biology Oct 2015Biological energy conversion in mitochondria is carried out by the membrane protein complexes of the respiratory chain and the mitochondrial ATP synthase in the inner... (Review)
Review
Biological energy conversion in mitochondria is carried out by the membrane protein complexes of the respiratory chain and the mitochondrial ATP synthase in the inner membrane cristae. Recent advances in electron cryomicroscopy have made possible new insights into the structural and functional arrangement of these complexes in the membrane, and how they change with age. This review places these advances in the context of what is already known, and discusses the fundamental questions that remain open but can now be approached.
Topics: Cellular Senescence; Cryoelectron Microscopy; Electron Transport; Membrane Proteins; Mitochondrial Membranes; Mitochondrial Proton-Translocating ATPases
PubMed: 26515107
DOI: 10.1186/s12915-015-0201-x -
FEBS Letters Aug 2014When taking up the gauntlet of studying membrane protein functionality, scientists are provided with a plethora of advantages, which can be exploited for the synthesis... (Review)
Review
When taking up the gauntlet of studying membrane protein functionality, scientists are provided with a plethora of advantages, which can be exploited for the synthesis of these difficult-to-express proteins by utilizing cell-free protein synthesis systems. Due to their hydrophobicity, membrane proteins have exceptional demands regarding their environment to ensure correct functionality. Thus, the challenge is to find the appropriate hydrophobic support that facilitates proper membrane protein folding. So far, various modes of membrane protein synthesis have been presented. Here, we summarize current state-of-the-art methodologies of membrane protein synthesis in biomimetic-supported systems. The correct folding and functionality of membrane proteins depend in many cases on their integration into a lipid bilayer and subsequent posttranslational modification. We highlight cell-free systems utilizing the advantages of biological membranes.
Topics: Animals; Biomimetic Materials; Cell Membrane; Cell-Free System; Humans; Membrane Proteins; Membranes, Artificial
PubMed: 24931371
DOI: 10.1016/j.febslet.2014.06.007 -
Biochemical Society Transactions Jun 2016Membrane proteins represent one of the most important targets for pharmaceutical companies. Unfortunately, technical limitations have long been a major hindrance in our... (Review)
Review
Membrane proteins represent one of the most important targets for pharmaceutical companies. Unfortunately, technical limitations have long been a major hindrance in our understanding of the function and structure of such proteins. Recent years have seen the refinement of classical approaches and the emergence of new technologies that have resulted in a significant step forward in the field of membrane protein research. This review summarizes some of the current techniques used for studying membrane proteins, with overall advantages and drawbacks for each method.
Topics: Bacteria; Eukaryota; Humans; Membrane Proteins; Membranes, Artificial; Methods
PubMed: 27284055
DOI: 10.1042/BST20160054 -
The Journal of Biological Chemistry Mar 2018Decades of work have contributed to our in-depth mechanistic understanding of soluble proteases, but much less is known about the catalytic mechanism of intramembrane... (Review)
Review
Decades of work have contributed to our in-depth mechanistic understanding of soluble proteases, but much less is known about the catalytic mechanism of intramembrane proteolysis due to inherent difficulties in both preparing and analyzing integral membrane enzymes and transmembrane substrates. New work from Naing tackles this challenge by examining the catalytic parameters of an aspartyl intramembrane protease homologous to the enzyme that cleaves amyloid precursor protein, finding that both chemistry and register contribute to specificity in substrate cleavage.
Topics: Amyloid; Aspartic Acid Proteases; Catalysis; Cell Membrane; Membrane Proteins; Proteolysis
PubMed: 29602877
DOI: 10.1074/jbc.H118.002210 -
Open Biology Dec 2021MLC1 is a membrane protein mainly expressed in astrocytes, and genetic mutations lead to the development of a leukodystrophy, megalencephalic leukoencephalopathy with...
MLC1 is a membrane protein mainly expressed in astrocytes, and genetic mutations lead to the development of a leukodystrophy, megalencephalic leukoencephalopathy with subcortical cysts disease. Currently, the biochemical properties of the MLC1 protein are largely unknown. In this study, we aimed to characterize the transmembrane (TM) topology and oligomeric nature of the MLC1 protein. Systematic immunofluorescence staining data revealed that the MLC1 protein has eight TM domains and that both the N- and C-terminus face the cytoplasm. We found that MLC1 can be purified as an oligomer and could form a trimeric complex in both detergent micelles and reconstituted proteoliposomes. Additionally, a single-molecule photobleaching experiment showed that MLC1 protein complexes could consist of three MLC1 monomers in the reconstituted proteoliposomes. These results can provide a basis for both the high-resolution structural determination and functional characterization of the MLC1 protein.
Topics: Amino Acid Sequence; Animals; COS Cells; Chlorocebus aethiops; Cytoplasm; HEK293 Cells; Humans; Membrane Proteins; Micelles; Protein Domains; Protein Multimerization; Proteolipids; Single Molecule Imaging
PubMed: 34847774
DOI: 10.1098/rsob.210103 -
Protein Science : a Publication of the... Dec 2020The question of how proteins manage to organize into a unique three-dimensional structure has been a major field of study since the first protein structures were... (Review)
Review
The question of how proteins manage to organize into a unique three-dimensional structure has been a major field of study since the first protein structures were determined. For membrane proteins, the question is made more complex because, unlike water-soluble proteins, the solvent is not homogenous or even unique. Each cell and organelle has a distinct lipid composition that can change in response to environmental stimuli. Thus, the study of membrane protein folding requires not only understanding how the unfolded chain navigates its way to the folded state, but also how changes in bilayer properties can affect that search. Here we review what we know so far about the impact of lipid composition on bilayer physical properties and how those properties can affect folding. A better understanding of the lipid bilayer and its effects on membrane protein folding is not only important for a theoretical understanding of the folding process, but can also have a practical impact on our ability to work with and design membrane proteins.
Topics: Cell Membrane; Lipid Bilayers; Membrane Proteins; Models, Molecular; Protein Folding; Protein Structure, Secondary
PubMed: 33058341
DOI: 10.1002/pro.3973 -
Biochemical Society Transactions Jun 2021Membrane proteins play vital roles in living organisms, serving as targets for most currently prescribed drugs. Membrane protein structural biology aims to provide... (Review)
Review
Membrane proteins play vital roles in living organisms, serving as targets for most currently prescribed drugs. Membrane protein structural biology aims to provide accurate structural information to understand their mechanisms of action. The advance of membrane protein structural biology has primarily relied on detergent-based methods over the past several decades. However, detergent-based approaches have significant drawbacks because detergents often damage the native protein-lipid interactions, which are often crucial for maintaining the natural structure and function of membrane proteins. Detergent-free methods recently have emerged as alternatives with a great promise, e.g. for high-resolution structure determinations of membrane proteins in their native cell membrane lipid environments. This minireview critically examines the current status of detergent-free methods by a comparative analysis of five groups of membrane protein structures determined using detergent-free and detergent-based methods. This analysis reveals that current detergent-free systems, such as the styrene-maleic acid lipid particles (SMALP), the diisobutyl maleic acid lipid particles (DIBMALP), and the cycloalkane-modified amphiphile polymer (CyclAPol) technologies are not better than detergent-based approaches in terms of maintenance of native cell membrane lipids on the transmembrane domain and high-resolution structure determination. However, another detergent-free technology, the native cell membrane nanoparticles (NCMN) system, demonstrated improved maintenance of native cell membrane lipids with the studied membrane proteins, and produced particles that were suitable for high-resolution structural analysis. The ongoing development of new membrane-active polymers and their optimization will facilitate the maturation of these new detergent-free systems.
Topics: Cell Membrane; Cryoelectron Microscopy; Detergents; Lipid Bilayers; Membrane Lipids; Membrane Proteins; Nanoparticles; Polymers; Protein Binding; Protein Conformation
PubMed: 34110369
DOI: 10.1042/BST20201080