Computational Design of Membrane Proteins

Pérez-Aguilar, José Manuel; Saven, Jeffery G.

doi:10.1016/j.str.2011.12.003

Cited by 33 publications

(31 citation statements)

References 88 publications

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“…The availability of a significant number of GPCR structures allowed us to seek sequence/structure correlations and general molecular determinants of conformational metastability across the class A GPCR family. Our present study focuses on suboptimal hydrogen bonds and van der Waals tertiary interactions, which represent two of the main physical forces stabilizing membrane protein structures (28,29,40,41).…”

Section: Resultsmentioning

confidence: 99%

“…In principle, by modeling protein structures at atomic resolution, computational structure-based design techniques can predict the energetics of hydrogen bonding and VDW interactions and engineer stabilizing mutations. However, such techniques have only been developed to design simple hydrophobic or cofactor-bound TM peptides (36-38) so far and have never been applied to large polytopic membrane proteins (28,(39)(40)(41).To address these limitations, we have developed a novel computational/experimental approach, which identifies metastable regions and designs stabilizing mutations in large membrane receptors. As a stringent proof of concept, we have applied our method to stabilize the WT beta1-adrenergic receptor (B1AR-WT) and a variant (B1AR-M23) previously thermostabilized by extensive scanning mutagenesis (26).…”

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confidence: 99%

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Naturally evolved G protein-coupled receptors adopt metastable conformations

Chen

Zhou

Fryszczyn

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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A wide range of membrane receptors signal through conformational changes, and the resulting protein conformational flexibility often hinders their structural studies. Because the determinants of membrane receptor conformational stability are still poorly understood, identifying a minimal set of perturbations stabilizing a membrane protein in a given conformation remains a major challenge in membrane protein structure determination. We present a novel approach integrating bioinformatics, computational design and experimental techniques that identifies and stabilizes metastable receptor regions. When applied to the beta1-adrenergic receptor, the method generated 13 novel receptor variants stabilized in the intended inactive state among which two exhibit an apparent thermostability higher than WT and M23 (a receptor variant previously stabilized by extensive scanning mutagenesis) by more than 30°C and 11°C, respectively. Targeted regions involve nonconserved unsatisfied polar residues or exhibit significant packing defects, features found in all class A G protein-coupled receptor structures. These findings suggest that natural G protein-coupled receptor sequences have evolved to be conformationally metastable through the design of suboptimal polar and van der Waals tertiary interactions. Given sufficiently accurate structural models, our approach should prove useful for designing stabilized variants of many uncharacterized membrane receptors.computational protein design | protein conformational stability | membrane protein modeling | signal transduction | synthetic biology M embrane proteins represent around 30% of currently sequenced genomes and are critical in the regulation of cell signaling and cell-cell communication (1, 2). When dysfunctional, however, these proteins can be responsible for serious diseases and constitute more than 60% of current drug targets. Despite their abundance and functional importance, membrane proteins are largely underrepresented in the Protein Data Bank, mainly due to technical difficulties in studying these proteins. A large fraction of these proteins such as G protein-coupled receptors (GPCRs) transduces signals across membranes through conformational changes (3-6). The resulting protein conformational heterogeneity and flexibility is a major factor hindering crystallization. An additional obstacle to structural determination is the poor stability of membrane proteins in detergent solution required by traditional crystallization approaches (7,8).In recent years, several approaches to stabilize membrane proteins have been explored (9). These methods include development and optimization of solubilizing detergents (10), reconstitution of proteins into lipidic bicelles (11, 12), cleavage of flexible protein regions (13-18), co-crystallization with stabilizing ligands, antibodies, and fusion with soluble proteins (e.g., T4 lysozyme) (13-15, 17, 19-23). Several of these modifications, however, disrupt important functional regions and interactions with either natural ligands or downstre...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Naturally evolved G protein-coupled receptors adopt metastable conformations

Chen

Zhou

Fryszczyn

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Previously, variants have been engineered of the potassium channel from Streptomyces lividans (KcsA) 19–21 and the transmembrane domain of the α1 subunit of the nicotinic acetylcholine receptor (nAChR), 22 with mutations ranging from 18% to 30% of the total number of residues. In our first version of the water-soluble analog of the human MUR, wsMUR-TM, 18% of the residues (53 out of 288) was mutated.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Computationally Designed Water-soluble Human μ-Opioid Receptor Variant Using Available Structural Information

et al. 2014

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Background The recent X-ray crystal structure of the murine μ opioid receptor (MUR) allowed us to reengineer a previously designed water-soluble variant of the transmembrane portion of the human MUR (wsMUR-TM). Methods The new variant of water soluble MUR (wsMUR-TM_v2) was engineered based upon the murine MUR crystal structure. This novel variant was expressed in E. coli and purified. The properties of the receptor were characterized and compared with those of wsMUR-TM. Results Seven residues originally included for mutation in the design of the wsMUR-TM, were reverted to their native identities. wsMUR-TM_v2 contains 16% mutations of the total sequence. It was overexpressed and purified with high yield. Although dimers and higher oligomers were observed to form over time, the wsMUR-TM_v2 stayed predominantly monomeric at concentrations as high as 7.5 mg/ml in buffer within a 2-month period. Its secondary structure was predominantly helical and comparable with those of both the original wsMUR-TM variant and the native MUR. The binding affinity of wsMUR-TM_v2 for naltrexone (Kd ~ 70 nM) was in close agreement with that for wsMUR-TM. The helical content of wsMUR-TM_v2 decreased cooperatively with increasing temperature, and the introduction of sucrose was able to stabilize the protein. Conclusions A novel functional wsMUR-TM_v2 with only 16% mutations was successfully engineered, expressed in E. coli and purified based on information from the crystal structure of murine MUR. This not only provides a novel alternative tool for MUR studies in solution conditions, but also offers valuable information for protein engineering and structure function relationships.

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“…Another interesting application in this vein is the creation of water soluble versions of membrane proteins [16,17]. Membrane proteins are an essential component of the cellular signaling network.…”

Section: Automated Protein Design: Radical Protein Engineering Bymentioning

confidence: 99%

Recent advances in automated protein design and its future challenges

Setiawan

Brender

Zhang

2018

Expert Opinion on Drug Discovery

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Introduction Protein function is determined by protein structure which is in turn determined by the corresponding protein sequence. If the rules that cause a protein to adopt a particular structure are understood, it should be possible to refine or even redefine the function of a protein by working backwards from the desired structure to the sequence. Automated protein design attempts to calculate the effects of mutations computationally with the goal of more radical or complex transformations than are accessible by experimental techniques. Areas covered The authors give a brief overview of the recent methodological advances in computer-aided protein design, showing how methodological choices affect final design and how automated protein design can be used to address problems considered beyond traditional protein engineering, including the creation of novel protein scaffolds for drug development. Also, the authors address specifically the future challenges in the development of automated protein design. Expert opinion Automated protein design holds potential as a protein engineering technique, particularly in cases where screening by combinatorial mutagenesis is problematic. Considering solubility and immunogenicity issues, automated protein design is initially more likely to make an impact as a research tool for exploring basic biology in drug discovery than in the design of protein biologics.

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Computational Design of Membrane Proteins

Cited by 33 publications

References 88 publications

Naturally evolved G protein-coupled receptors adopt metastable conformations

Naturally evolved G protein-coupled receptors adopt metastable conformations

Characterization of a Computationally Designed Water-soluble Human μ-Opioid Receptor Variant Using Available Structural Information

Recent advances in automated protein design and its future challenges

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