Abstract:
:Chemical synthesis is a powerful method for precise modification of the structural and electronic properties of proteins. The difficulties in the synthesis and purification of peptides containing transmembrane segments have presented obstacles to the chemical synthesis of integral membrane proteins. Here, we present a modular strategy for the semisynthesis of integral membrane proteins in which solid-phase peptide synthesis is limited to the region of interest, while the rest of the protein is obtained by recombinant means. This modular strategy considerably simplifies the synthesis and purification steps that have previously hindered the chemical synthesis of integral membrane proteins. We develop a SUMO fusion and proteolysis approach for obtaining the N-terminal cysteine containing membrane-spanning peptides required for the semisynthesis. We demonstrate the feasibility of the modular approach by the semisynthesis of full-length KcsA K(+) channels in which only regions of interest, such as the selectivity filter or the pore helix, are obtained by chemical synthesis. The modular approach is used to investigate the hydrogen bond interactions of a tryptophan residue in the pore helix, tryptophan 68, by substituting it with the isosteric analogue, beta-(3-benzothienyl)-l-alanine (3BT). A functional analysis of the 3BT mutant channels indicates that the K(+) conduction and selectivity of the 3BT mutant channels are similar to those of the wild type, but the mutant channels show a 3-fold increase in Rb(+) conduction. These results suggest that the hydrogen bond interactions of tryptophan 68 are essential for optimizing the selectivity filter for K(+) conduction over Rb(+) conduction.
journal_name
ACS Chem Bioljournal_title
ACS chemical biologyauthors
Komarov AG,Linn KM,Devereaux JJ,Valiyaveetil FIdoi
10.1021/cb900210rsubject
Has Abstractpub_date
2009-12-18 00:00:00pages
1029-38issue
12eissn
1554-8929issn
1554-8937journal_volume
4pub_type
杂志文章abstract::A biophysical understanding of the mechanistic, chemical, and physical origins underlying antibiotic action and resistance is vital to the discovery of novel therapeutics and the development of strategies to combat the growing emergence of antibiotic resistance. The site-specific introduction of stable-isotope labels ...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/acschembio.9b01054
更新日期:2020-05-15 00:00:00
abstract::The endoplasmic reticulum (ER) is the initial site of biogenesis of secretory pathway proteins, including proteins localized to the ER, Golgi, lysosomes, intracellular vesicles, plasma membrane, and extracellular compartments. Proteins within the secretory pathway contain a high abundance of disulfide bonds to protect...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/acschembio.9b01014
更新日期:2020-02-21 00:00:00
abstract::Vitamin A serves essential functions in mammalian biology as a signaling molecule and chromophore. This lipid can be synthesized from more than 50 putative dietary provitamin A precursor molecules which contain at least one unsubstituted β-ionone ring. We here scrutinized the enzymatic properties and substrate specifi...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/acschembio.8b00290
更新日期:2018-08-17 00:00:00
abstract::Though phenotypic and target-based high-throughput screening approaches have been employed to discover new antibiotics, the identification of promising therapeutic candidates remains challenging. Each approach provides different information, and understanding their results can provide hypotheses for a mechanism of act...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/acschembio.7b00468
更新日期:2017-09-15 00:00:00
abstract::The Bcl-2 family of proteins is crucial for apoptosis regulation. Members of this family insert through a specific C-terminal anchoring transmembrane domain (TMD) in the mitochondrial outer membrane where they hierarchically interact to determine cell fate. While the mitochondrial membrane has been proposed to activel...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/cb5002679
更新日期:2014-08-15 00:00:00
abstract::A major risk for patients having estrogen receptor α (ERα)-positive breast cancer is the recurrence of drug-resistant metastases after initial successful treatment with endocrine therapies. Recent studies have implicated a number of activating mutations in the ligand-binding domain of ERα that stabilize the agonist co...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/acschembio.8b00877
更新日期:2018-12-21 00:00:00
abstract::Acute and specific sensing of diatomic gas molecules is an essential facet of biological signaling. Heme nitric oxide/oxygen binding (H-NOX) proteins are a family of gas sensors found in diverse classes of bacteria and eukaryotes. The most commonly characterized bacterial H-NOX domains are from facultative anaerobes a...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/acschembio.6b00431
更新日期:2016-08-19 00:00:00
abstract::Thermodynamics and kinetics of protein-ligand binding are both important aspects for the design of novel drug molecules. Presently, thermodynamic data are collected with isothermal titration calorimetry, while kinetic data are mostly derived from surface plasmon resonance. The new method of kinITC provides both thermo...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/acschembio.9b00895
更新日期:2020-03-20 00:00:00
abstract::Reporter gene assays (RGAs) are commonly used to measure biological pathway modulation by small molecules. Understanding how such compounds interact with the reporter enzyme is critical to accurately interpret RGA results. To improve our understanding of reporter enzymes and to develop optimal RGA systems, we investig...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/cb3007264
更新日期:2013-05-17 00:00:00
abstract::The rational design of environmentally sensitive dyes with superior properties is critical for elucidating the fundamental biological processes and understanding the biophysical behavior of cell membranes. In this study, a novel group of fluorene-based push-pull probes was developed for imaging membrane lipids. The de...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/acschembio.7b00658
更新日期:2017-12-15 00:00:00
abstract::Many oncogenic mutants of the tumor suppressor p53 are conformationally unstable, including the frequently occurring Y220C mutant. We have previously developed several small-molecule stabilizers of this mutant. One of these molecules, PhiKan083, 1-(9-ethyl-9H-carbazole-3-yl)-N-methylmethanamine, binds to a mutation-in...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/acschembio.6b00315
更新日期:2016-08-19 00:00:00
abstract::Labeling of virus opens new pathways for the understanding of viruses themselves and facilitates the utilization of viruses in modern biology, medicine, and materials. Based on the characteristic that viruses hijack their host cellular machineries to survive and reproduce themselves, a host-cell-assisted strategy is p...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/cb2001878
更新日期:2012-04-20 00:00:00
abstract::Cell-penetrating peptides (CPPs) are capable of delivering membrane-impermeable cargoes (including small molecules, peptides, proteins, nucleic acids, and nanoparticles) into the cytosol of mammalian cells and have the potential to revolutionize biomedical research and drug discovery. However, the mechanism of action ...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/acschembio.0c00478
更新日期:2020-09-18 00:00:00
abstract::Bacteria use small molecules to assess the density and identity of nearby organisms and formulate a response. This process, called quorum sensing (QS), commonly regulates bioluminescence, biofilm formation, and virulence. Vibrio harveyi have three described QS circuits. Each involves the synthesis of a molecule that r...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/cb300215t
更新日期:2012-08-17 00:00:00
abstract::The display and analysis of proteins expressed on biological surfaces has become an attractive tool for the study of molecular interactions in enzymology, protein engineering, and high-throughput screening. Among the growing number of established display systems, retroviruses offer a unique and fully mammalian platfor...
journal_title:ACS chemical biology
pub_type: 杂志文章,评审
doi:10.1021/cb100285n
更新日期:2011-01-21 00:00:00
abstract::Membrane proteins perform their functions within or on the lipid membrane, and lipid compositions are known to affect membrane protein integration and activity. Recently, the geometric aspect of membrane curvature was shown to play an important role in membrane protein behavior. Certain membrane proteins are known to ...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/acschembio.5b00107
更新日期:2015-07-17 00:00:00
abstract::The human ClpP proteolytic complex (HsClpP) is a serine protease located in the mitochondrial matrix and participates in the maintenance of the mitochondrial proteome among other cellular functions. HsClpP typically forms a multimeric complex with the AAA+ protein unfoldase HsClpX. Notably, compared to that of normal,...
journal_title:ACS chemical biology
pub_type: 杂志文章,评审
doi:10.1021/acschembio.9b00347
更新日期:2019-11-15 00:00:00
abstract::We report here an isatin derivative 45 (ID45) against coxsackievirus B3 (CVB3) replication, which was synthesized based on a high-throughput screen of a unique natural product library. ID45 showed the most potent anti-CVB3 activity among the four synthesized compounds. Treatment of cells with ID45 before or after infe...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/cb400775z
更新日期:2014-04-18 00:00:00
abstract::RNA modifications play important roles in RNA structures and regulation of gene expression and translation. We report the first RNA modification on the phosphate, the RNA phosphorothioate (PS) modification, discovered in both prokaryotes and eukaryotes. The PS modification is also first reported on nucleic acids of eu...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/acschembio.0c00163
更新日期:2020-06-19 00:00:00
abstract::Extracellular expression of heat shock protein 90 (eHsp90) by tumor cells is correlated with malignancy. Development of small molecule probes that can detect eHsp90 in vivo may therefore have utility in the early detection of malignancy. We synthesized a cell impermeable far-red fluorophore-tagged Hsp90 inhibitor to t...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/acschembio.7b00006
更新日期:2017-04-21 00:00:00
abstract::3-Oxo-β-sultams are four-membered ring ambident electrophiles that can react with nucleophiles either at the carbonyl carbon or at the sulfonyl sulfur atoms, and that have been reported to inhibit serine hydrolases via acylation of the active-site serine residue. We have developed a panel of 3-oxo-β-sultam inhibitors ...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/acschembio.0c00090
更新日期:2020-04-17 00:00:00
abstract::The number of bacterial strains that are resistant against antibiotics increased dramatically during the past decades. This fact stresses the urgent need for the development of new antibacterial agents with novel modes of action targeting essential enzymes such as RNA polymerase (RNAP). Bacterial RNAP is a large multi...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/cb3005758
更新日期:2013-04-19 00:00:00
abstract::Beta-peptides (beta-amino acid oligomers) that mimic the amphiphilic, helical, and cationic properties of natural antimicrobial peptides have previously been shown to display antifungal activity against planktonic Candida albicans cells. Beta-peptides offer several advantages over conventional peptides composed of alp...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/cb900093r
更新日期:2009-07-17 00:00:00
abstract::Apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii, the causative agents of severe malaria and toxoplasmosis, respectively, undergo several critical developmental transitions during their lifecycle. Most important for human pathogenesis is the asexual cycle, in which parasites undergo rounds...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/cb501004q
更新日期:2015-04-17 00:00:00
abstract::The gibberellin (GA) phytohormones are produced not only by plants but also by fungi and bacteria. Previous characterization of a cytochrome P450 (CYP)-rich GA biosynthetic operon found in many symbiotic, nitrogen-fixing rhizobia led to the elucidation of bacterial GA biosynthesis and implicated GA9 as the final produ...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/acschembio.6b01038
更新日期:2017-04-21 00:00:00
abstract::A DNA-encoded macrocyclic peptide library was designed and synthesized with 2.4 × 1012 members composed of 4-20 natural and non-natural amino acids. Affinity-based selection was performed against two therapeutic targets, VHL and RSV N protein. On the basis of selection data, some peptides were selected for resynthesis...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/acschembio.7b00852
更新日期:2018-01-19 00:00:00
abstract::Regulators of G protein signaling (RGS) proteins are key players in regulating signaling via G protein-coupled receptors. RGS proteins directly bind to the Gα-subunits of activated heterotrimeric G-proteins, and accelerate the rate of GTP hydrolysis, thereby rapidly deactivating G-proteins. Using atomistic simulations...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/cb400568g
更新日期:2013-12-20 00:00:00
abstract::APOBEC3G is a single-stranded DNA cytosine deaminase that comprises part of the innate immune response to viruses and transposons. Although APOBEC3G is the prototype for understanding the larger mammalian polynucleotide deaminase family, no specific chemical inhibitors exist to modulate its activity. High-throughput s...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/cb200440y
更新日期:2012-03-16 00:00:00
abstract::How primitive enzymes emerged from a primordial pool remains a fundamental unanswered question with important practical implications in synthetic biology. Here we show that a de novo evolved ATP binding protein, selected solely on the basis of its ability to bind ATP, mediates the regiospecific hydrolysis of ATP to AD...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/cb900109w
更新日期:2009-08-21 00:00:00
abstract::Recent advances in understanding the relevance of noncoding RNA (ncRNA) to disease have increased interest in drugging ncRNA with small molecules. The recent discovery of ribocil, a structurally distinct synthetic mimic of the natural ligand of the flavin mononucleotide (FMN) riboswitch, has revealed the potential che...
journal_title:ACS chemical biology
pub_type: 杂志文章
doi:10.1021/acschembio.7b01013
更新日期:2018-03-16 00:00:00