RESUMEN
We present the first demonstration of ligand-induced conformational changes in a biological molecule, a protein, by sum-frequency generation (SFG). Constructs of KRasG12D protein were prepared by selectively deuterating residues of a single amino acid type using isotope-labeled amino acids and cell-free protein synthesis. By attaching labeled protein to a supported bilayer membrane via a His-tag to Ni-NTA-bearing lipids, we ensured that single layers of ordered molecules were formed while preserving the protein's native structure. Exceptionally large SFG amide I signals were produced in both labeled and unlabeled proteins, demonstrating a high degree of orientational order upon attachment to the bilayer. Deuterated protein also produced SFG signals in the CDx spectral region, which were not present in the unlabeled protein. The CDx signals were measured before and after binding a peptide inhibitor, KRpep-2d, revealing shifts in SFG intensity due to conformational changes at the labeled sites. In particular, peaks associated with CDx stretching vibrations for alanine, valine, and glycine changed substantially in amplitude upon inhibitor binding. By inspection of the crystal structure, these three residues are uniquely colocated on the protein surface in and near the nucleotide binding site, which is in allosteric communication with the site of peptide inhibitor binding, suggesting an approach to identify a ligand's binding site. The technique offers a highly sensitive, nonperturbative method of mapping ligand-induced conformational changes and allosteric networks in biological molecules for studies of the relationship between structure and function and mechanisms of action in drug discovery.
RESUMEN
Global substitution of leucine for analogues containing CH2F instead of methyl groups delivers proteins with multiple sites for monitoring by 19F nuclear magnetic resonance (NMR) spectroscopy. The 19 kDa Escherichia coli peptidyl-prolyl cis-trans isomerase B (PpiB) was prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5'-difluoroleucine. The stability of the samples toward thermal denaturation was little altered compared to the wild-type protein. 19F nuclear magnetic resonance (NMR) spectra showed large chemical shift dispersions between 6 and 17 ppm. The 19F chemical shifts correlate with the three-bond 1H-19F couplings (3JHF), providing the first experimental verification of the γ-gauche effect predicted by [Feeney, J. J. Am. Chem. Soc. 1996, 118, 8700-8706] and establishing the effect as the predominant determinant of the 19F chemical shifts of CH2F groups. Individual CH2F groups can be confined to single rotameric states by the protein environment, but most CH2F groups exchange between different rotamers at a rate that is fast on the NMR chemical shift scale. Interactions between fluorine atoms in 5,5'-difluoroleucine bias the CH2F rotamers in agreement with results obtained previously for 1,3-difluoropropane. The sensitivity of the 19F chemical shift to the rotameric state of the CH2F groups potentially renders them particularly sensitive for detecting allosteric effects.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Isomerasa de Peptidilprolil , Isomerasa de Peptidilprolil/metabolismo , Isomerasa de Peptidilprolil/química , Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/enzimología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ligandos , Resonancia Magnética Nuclear Biomolecular/métodos , Leucina/química , Leucina/metabolismo , Leucina/análogos & derivados , Flúor/químicaRESUMEN
(2S,3S)-4-Fluorovaline (FVal) is an analogue of valine, where a single CH3 group is substituted by a CH2F group. In the absence of valine, E. coli valyl-tRNA synthetase uses FVal as a substitute, enabling the production of proteins uniformly labeled with FVal. Here, we describe the production and analysis of E. coli peptidyl-prolyl isomerase B where all 16 valine residues have been replaced by FVal synthesized with a 13C-labeled CH2F group. Although the melting temperature is lower by about 11 °C relative to the wild-type protein, the three-dimensional protein structure is almost completely conserved, as shown by X-ray crystallography. The CH2F groups invariably populate staggered rotamers. Most CH2F groups populate two different rotamers. The increased space requirement of fluorine versus hydrogen does not prohibit rotamers that position fluorine next to a backbone carbonyl carbon. 19F NMR spectra show a signal dispersion over 25 ppm. The most high-field shifted 19F resonances correlate with large 3JHF coupling constants, confirming the impact of the γ-gauche effect on the signal dispersion. The present work is the second experimental verification of the effect and extends its validity to fluorovaline. The abundance of valine in proteins and structural conservation with FVal renders this valine analogue attractive for probing proteins by 19F NMR spectroscopy.
Asunto(s)
Escherichia coli , Isomerasa de Peptidilprolil , Valina , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/enzimología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Modelos Moleculares , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/metabolismo , Isomerasa de Peptidilprolil/genética , Conformación Proteica , Valina/química , Valina/metabolismoRESUMEN
Proteins produced with leucine analogues, where CH2F groups substitute specific methyl groups, can readily be probed by 19F NMR spectroscopy. As CF and CH groups are similar in hydrophobicity and size, fluorinated leucines are expected to cause minimal structural perturbation, but the impact of fluorine on the rotational freedom of CH2F groups is unclear. We present high-resolution crystal structures of Escherichia coli peptidyl-prolyl cis-trans isomerase B (PpiB) prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5'-difluoroleucine. Apart from the fluorinated leucine residues, the structures show complete structural conservation of the protein backbone and the amino acid side chains except for a single isoleucine side chain located next to a fluorine atom in the hydrophobic core of the protein. The carbon skeletons of the fluorinated leucine side chains are also mostly conserved. The CH2F groups show a strong preference for staggered rotamers and often appear locked into single rotamers. Substitution of leucine CH3 groups for CH2F groups is thus readily tolerated in the three-dimensional (3D) structure of a protein, and the rotation of CH2F groups can be halted at cryogenic temperatures.
Asunto(s)
Leucina , Leucina/química , Escherichia coli/metabolismo , Conformación Proteica , Modelos Moleculares , Cristalografía por Rayos X , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismoRESUMEN
The substitution of a single hydrogen atom in a protein by fluorine yields a site-specific probe for sensitive detection by 19F nuclear magnetic resonance (NMR) spectroscopy, where the absence of background signal from the protein facilitates the detection of minor conformational species. We developed genetic encoding systems for the site-selective incorporation of 4-fluorotryptophan, 5-fluorotryptophan, 6-fluorotryptophan, and 7-fluorotryptophan in response to an amber stop codon and used them to investigate conformational heterogeneity in a designed amino acid binding protein and in flaviviral NS2B-NS3 proteases. These proteases have been shown to present variable conformations in X-ray crystal structures, including flips of the indole side chains of tryptophan residues. The 19F NMR spectra of different fluorotryptophan isomers installed at the conserved site of Trp83 indicate that the indole ring flip is common in flaviviral NS2B-NS3 proteases in the apo state and suppressed by an active-site inhibitor.
Asunto(s)
Conformación Proteica , Triptófano , Triptófano/química , Triptófano/análogos & derivados , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Flúor/química , Proteínas/químicaRESUMEN
Paramagnetic chemical probes have been used in electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopy for more than four decades. Recent years witnessed a great increase in the variety of probes for the study of biological macromolecules (proteins, nucleic acids, and oligosaccharides). This Review aims to provide a comprehensive overview of the existing paramagnetic chemical probes, including chemical synthetic approaches, functional properties, and selected applications. Recent developments have seen, in particular, a rapid expansion of the range of lanthanoid probes with anisotropic magnetic susceptibilities for the generation of structural restraints based on residual dipolar couplings and pseudocontact shifts in solution and solid state NMR spectroscopy, mostly for protein studies. Also many new isotropic paramagnetic probes, suitable for NMR measurements of paramagnetic relaxation enhancements, as well as EPR spectroscopic studies (in particular double resonance techniques) have been developed and employed to investigate biological macromolecules. Notwithstanding the large number of reported probes, only few have found broad application and further development of probes for dedicated applications is foreseen.
Asunto(s)
Ácidos Nucleicos , Proteínas , Espectroscopía de Resonancia por Spin del Electrón , Espectroscopía de Resonancia Magnética , Resonancia Magnética Nuclear Biomolecular/métodos , Ácidos Nucleicos/química , Oligosacáridos , Proteínas/químicaRESUMEN
DFT calculations indicate that the 19F chemical shifts of aromatic rings containing single fluorine substituents are sensitive to the electric fields and electric field gradients at the position of the fluorine atom. The present work explores whether long-range structure restraints can be gained from changes in 19F chemical shifts following mutations of charged to uncharged residues. 19F chemical shifts of fluorotryptophan residues were measured in two different proteins, GB1 and the NT* domain, following mutations of single asparagine residues to aspartic acid. Four different versions of fluorotryptophan were investigated, including 4-, 5-, 6-, and 7-fluorotryptophan, which were simultaneously installed by cell-free protein synthesis using 4-, 5-, 6-, and 7-fluoroindole as precursors for the tryptophan synthase present in the S30 extract. For comparison, the 1H chemical shifts of the corresponding nonfluorinated protein mutants produced with 13C-labeled tryptophan were also measured. The results show that the 19F chemical shifts respond more sensitively to the charge mutations than the 1H chemical shifts in the nonfluorinated references, but the chemical shift changes were much smaller than predicted by DFT calculations of fluoroindoles in the electric field of a partial charge in vacuum, indicating comprehensive dielectric shielding by water and protein. No straightforward correlation with the location of the charge mutation could be established.
Asunto(s)
Flúor , Espectroscopía de Resonancia Magnética/métodos , Electricidad Estática , Flúor/químicaRESUMEN
Cell-free protein synthesis using eCells allows production of amino acids from inexpensive 13C-labelled precursors. We show that the metabolic pathway converting pyruvate, glucose and erythrose into aromatic amino acids is maintained in eCells. Judicious choice of 13C-labelled starting material leads to proteins, where the sidechains of aromatic amino acids display [13C,1H]-HSQC cross-peaks free of one-bond 13C-13C couplings. Selective 13C-labelling of tyrosine and phenylalanine residues is achieved simply by using different compositions of the reaction buffers.
Asunto(s)
Aminoácidos Aromáticos , Proteínas , Resonancia Magnética Nuclear Biomolecular , Proteínas/química , Aminoácidos Aromáticos/química , Aminoácidos/química , Tirosina/químicaRESUMEN
The COVID-19 pandemic continues to be a public health threat. Multiple mutations in the spike protein of emerging variants of SARS-CoV-2 appear to impact on the effectiveness of available vaccines. Specific antiviral agents are keenly anticipated but their efficacy may also be compromised in emerging variants. One of the most attractive coronaviral drug targets is the main protease (Mpro). A promising Mpro inhibitor of clinical relevance is the peptidomimetic nirmatrelvir (PF-07321332). We expressed Mpro of six SARS-CoV-2 lineages (C.37 Lambda, B.1.1.318, B.1.2, B.1.351 Beta, B.1.1.529 Omicron, P.2 Zeta), each of which carries a strongly prevalent missense mutation (G15S, T21I, L89F, K90R, P132H, L205V). Enzyme kinetics reveal that these Mpro variants are catalytically competent to a similar degree as the wildtype. We show that nirmatrelvir has similar potency against the variants as the wildtype. Our in vitro data suggest that the efficacy of the specific Mpro inhibitor nirmatrelvir is not compromised in current COVID-19 variants.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Antivirales/farmacología , Antivirales/uso terapéutico , Humanos , Lactamas , Leucina , Nitrilos , Pandemias/prevención & control , Péptido Hidrolasas , Prolina , Inhibidores de Proteasas , SARS-CoV-2/genéticaRESUMEN
Efficient syntheses of fluorinated leucines, valines and alanines are described. The synthetic routes provide expedient access to various 13C/15N/D isotopologues requiring solely readily available and inexpensive isotope containing reagents such as NaBD4, carbon-13C dioxide and sodium azide-1-15N. The lightly fluorinated leucines and valines were found to be good substrates for cell-free protein expression and even 3-fluoroalanine, which is highly toxic to bacteria in vivo, could be incorporated into proteins this way. 19F-NMR spectra of the protein GB1 produced with these amino acids showed large chemical shift dispersions. Particularly high incorporation yields and clean 19F-NMR spectra were obtained for GB1 produced with valine residues, which had been synthesized with a single fluorine substituting a hydrogen stereospecifically in one of the methyl groups.
Asunto(s)
Alanina , Valina , Flúor/química , Leucina/química , Espectroscopía de Resonancia Magnética , Proteínas/química , Valina/químicaRESUMEN
The measurement of distances in proteins can be challenging in the 5-20 Å range, which is outside those accessible through conventional NMR and EPR methods. Recently it was demonstrated that distances in this range could be measured between a nitroxide as a paramagnetic spin label and a nearby fluorine atom (19F) as a nuclear spin label using high-field (W-band/3.4 T) ENDOR spectroscopy. Here we show that such measurements can also be performed using a gadolinium ion (Gd3+) as the paramagnetic tag. Gd3+ has two advantages. (i) A greater electronic spin (S = 7/2) and fast electronic spin-lattice (T1) relaxation, improving sensitivity by allowing data to be collected at lower temperatures. (ii) A narrow EPR signal for the -½ â ½ transition, and therefore no orientation selection artefacts. Signal intensities can be further enhanced by using a trifluoromethyl (C19F3) group instead of a single 19F atom. Using the protein calbindin D9k with a Ca2+ ion replaced by a Gd3+ ion and a trifluoromethylphenylalanine in position 50, we show that distances up to about 10 Å can be readily measured. Longer distances proved more difficult to measure due to variable electronic TM relaxation rates, which lead to broader Lorentzian ENDOR lineshapes. Gd3+ complexes (Gd3+ tags), which reliably display longer TM times, allow longer distances to be measured (8-16 Å). We also provide preliminary evidence that the intensity of ENDOR signals follows the predicted 1/r6 dependence, indicating that distances r > 20 Å can be measured by this method.
Asunto(s)
Gadolinio , Proteínas , Espectroscopía de Resonancia por Spin del Electrón/métodos , Marcadores de Spin , Proteínas/química , Gadolinio/química , Espectroscopía de Resonancia MagnéticaRESUMEN
Cyanopyridylalanines are non-canonical amino acids that react with aminothiol compounds under physiological conditions in a biocompatible manner without requiring added catalyst. Here we present newly developed aminoacyl-tRNA synthetases for genetic encoding of meta- and para-cyanopyridylalanine to enable the site-specific attachment of a wide range of different functionalities. The outstanding utility of the cyanopyridine moiety is demonstrated by examples of i) post-translational functionalization of proteins, ii)â in-cell macrocyclization of peptides and proteins, and iii)â protein stapling. The biocompatible nature of the protein ligation chemistry enabled by the cyanopyridylalanine amino acid opens a new path to specific in vivo protein modifications in complex biological environments.
Asunto(s)
Aminoacil-ARNt Sintetasas , Nitrilos , Aminas , Aminoácidos/química , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Proteínas/química , Compuestos de SulfhidriloRESUMEN
Trimethylsilyl (TMS) groups present outstanding NMR probes of biological macromolecules as they produce intense singlets in 1H NMR spectra near 0 ppm, where few other proton resonances occur. We report a system for genetic encoding of N6-(((trimethylsilyl)methoxy)carbonyl)-l-lysine (TMSK) for site-specific incorporation into proteins. The system is based on pyrrolysyl-tRNA synthetase mutants, which deliver proteins with high yield and purity in vivo and in cell-free protein synthesis. As the TMS signal can readily be identified in 1D 1H NMR spectra of high-molecular weight systems without the need of isotopic labeling, TMSK delivers an excellent site-specific NMR probe for the study of protein structure and function, which is both inexpensive and convenient. We demonstrate the utility of TMSK to detect ligand binding, measure the rate of conformational change, and assess protein dimerization by paramagnetic relaxation enhancement. In addition, we present a system for dual incorporation of two different unnatural amino acids (TMSK and O-tert-butyl-tyrosine) in the same protein in quantities sufficient for NMR spectroscopy. Close proximity of the TMS and tert-butyl groups was readily detected by nuclear Overhauser effects.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Lisina/química , Resonancia Magnética Nuclear Biomolecular , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Ligandos , Lisina/análogos & derivados , Lisina/genética , Modelos Moleculares , Estructura Molecular , Peso Molecular , Mutación , Unión ProteicaRESUMEN
Fluorine atoms are known to display scalar 19F-19F couplings in nuclear magnetic resonance (NMR) spectra when they are sufficiently close in space for nonbonding orbitals to overlap. We show that fluorinated noncanonical amino acids positioned in the hydrophobic core or on the surface of a protein can be linked by scalar through-space 19F-19F (TSJFF) couplings even if the 19F spins are in the time average separated by more than the van der Waals distance. Using two different aromatic amino acids featuring CF3 groups, O-trifluoromethyl-tyrosine and 4-trifluoromethyl-phenylalanine, we show that 19F-19F TOCSY experiments are sufficiently sensitive to detect TSJFF couplings between 2.5 and 5 Hz in the 19 kDa protein PpiB measured on a two-channel 400 MHz NMR spectrometer with a regular room temperature probe. A quantitative J evolution experiment enables the measurement of TSJFF coupling constants that are up to five times smaller than the 19F NMR line width. In addition, a new aminoacyl-tRNA synthetase was identified for genetic encoding of N6-(trifluoroacetyl)-l-lysine (TFA-Lys) and 19F-19F TOCSY peaks were observed between two TFA-Lys residues incorporated into the proteins AncCDT-1 and mRFP despite high solvent exposure and flexibility of the TFA-Lys side chains. With the ready availability of systems for site-specific incorporation of fluorinated amino acids into proteins by genetic encoding, 19F-19F interactions offer a straightforward way to probe the spatial proximity of selected sites without any assignments of 1H NMR resonances.
Asunto(s)
Aminoácidos/análisis , Isomerasa de Peptidilprolil/química , Flúor , Halogenación , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Resonancia Magnética Nuclear BiomolecularRESUMEN
The selenol group of selenocysteine is much more nucleophilic than the thiol group of cysteine. Selenocysteine residues in proteins thus offer reactive points for rapid post-translational modification. Herein, we show that selenoproteins can be expressed in high yield and purity by cell-free protein synthesis by global substitution of cysteine by selenocysteine. Complete alkylation of solvent-exposed selenocysteine residues was achieved in 10 minutes with 4-chloromethylene dipicolinic acid (4Cl-MDPA) under conditions that left cysteine residues unchanged even after overnight incubation. GdIII -GdIII distances measured by double electron-electron resonance (DEER) experiments of maltose binding protein (MBP) containing two selenocysteine residues tagged with 4Cl-MDPA-GdIII were indistinguishable from GdIII -GdIII distances measured of MBP containing cysteine reacted with 4Br-MDPA tags.
Asunto(s)
Proteínas de Unión a Maltosa/análisis , Ácidos Picolínicos/química , Selenoproteínas/química , Estructura Molecular , Selenoproteínas/síntesis químicaRESUMEN
A lanthanide-binding tag site-specifically attached to a protein presents a tool to probe the protein by multiple spectroscopic techniques, including nuclear magnetic resonance, electron paramagnetic resonance and time-resolved luminescence spectroscopy. Here a new stable chiral LnIII tag, referred to as C12, is presented for spontaneous and quantitative reaction with a cysteine residue to generate a stable thioether bond. The synthetic protocol of the tag is relatively straightforward, and the tag is stable for storage and shipping. It displays greatly enhanced reactivity towards selenocysteine, opening a route towards selective tagging of selenocysteine in proteins containing cysteine residues. Loaded with TbIII or TmIII ions, the C12 tag readily generates pseudocontact shifts (PCS) in protein NMR spectra. It produces a relatively rigid tether between lanthanide and protein, which is beneficial for interpretation of the PCSs by single magnetic susceptibility anisotropy tensors, and it is suitable for measuring distance distributions in double electron-electron resonance experiments. Upon reaction with cysteine or other thiol compounds, the TbIII complex exhibits a 100-fold enhancement in luminescence quantum yield, affording a highly sensitive turn-on luminescence probe for time-resolved FRET assays and enzyme reaction monitoring.
Asunto(s)
Elementos de la Serie de los Lantanoides , Cisteína , Luminiscencia , Resonancia Magnética Nuclear Biomolecular , ProteínasRESUMEN
Specific anti-coronaviral drugs complementing available vaccines are urgently needed to fight the COVID-19 pandemic. Given its high conservation across the betacoronavirus genus and dissimilarity to human proteases, the SARS-CoV-2 main protease (Mpro) is an attractive drug target. SARS-CoV-2 Mpro inhibitors have been developed at unprecedented speed, most of them being substrate-derived peptidomimetics with cysteine-modifying warheads. In this study, Mpro has proven resistant towards the identification of high-affinity short substrate-derived peptides and peptidomimetics without warheads. 20 cyclic and linear substrate analogues bearing natural and unnatural residues, which were predicted by computational modelling to bind with high affinity and designed to establish structure-activity relationships, displayed no inhibitory activity at concentrations as high as 100 µM. Only a long linear peptide covering residues P6 to P5' displayed moderate inhibition (Ki = 57 µM). Our detailed findings will inform current and future drug discovery campaigns targeting Mpro.
Asunto(s)
COVID-19/patología , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Inhibidores de Proteasas/química , SARS-CoV-2/enzimología , COVID-19/virología , Proteasas 3C de Coronavirus/metabolismo , Cisteína/química , Cisteína/metabolismo , Humanos , Lactamas/química , Lactamas/metabolismo , Leucina/química , Leucina/metabolismo , Nitrilos/química , Nitrilos/metabolismo , Péptidos/química , Péptidos/metabolismo , Peptidomiméticos/química , Peptidomiméticos/metabolismo , Prolina/química , Prolina/metabolismo , Inhibidores de Proteasas/metabolismo , SARS-CoV-2/aislamiento & purificación , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Synthesis of indoles labeled with 13C-1H and 13C-19F spin pairs is described. All syntheses utilize inexpensive carbon-13C dioxide as the 13C isotope source. Ruthenium-mediated ring-closing metathesis is the key step in construction of the 13C containing indole carbocycle. Fluorine is introduced via electrophilic fluorination at the 7-position and via palladium-mediated cross-coupling at the 4-position. Indole and fluoroindoles are viable tryptophan precursors for in vivo protein expression. We show that they are viable also in in vitro protein synthesis using standard E. coli S30 extracts. Incorporation of the synthesized 13C-1H and 13C-19F spin pair labeled tryptophans into proteins enables high-resolution and high-sensitivity nuclear magnetic resonance (NMR) spectroscopy.
Asunto(s)
Indoles/química , Resonancia Magnética Nuclear Biomolecular , Triptófano/análisis , Isótopos de Carbono , Deuterio , Flúor , Indoles/síntesis químicaRESUMEN
SF5Phe, para-pentafluorosulfanyl phenylalanine, is an unnatural amino acid with extreme physicochemical properties, which is stable in physiological conditions. Here we present newly developed aminoacyl-tRNA synthetases that enable genetic encoding of SF5Phe for site-specific incorporation into proteins in high yields. Owing to the SF5 moiety's dichotomy of strong polarity and high hydrophobicity, the unnatural amino acid forms specific and strong interactions in proteins. The potential of SF5Phe in protein research is illustrated by (i) increasing the binding affinity of a consensus pentapeptide motif toward the ß subunit of Escherichia coli DNA polymerase III holoenzyme by mutation of a phenylalanine to a SF5Phe residue, (ii) site-specifically adhering ß-cyclodextrin to the surface of ubiquitin, and (iii) selective detection of 19F-19F nuclear Overhauser effects in the Escherichia coli peptidyl-prolyl cis/trans-isomerase B following mutation of two phenylalanine residues in the core of the protein to SF5Phe. With increasing use of the SF5 moiety in pharmaceutical chemistry, this general method of functionalizing proteins with SF5 groups opens unique opportunities for structural biology and in vivo studies.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , ADN Polimerasa III/metabolismo , Fluorocarburos/química , Fenilalanina/química , Aminoacil-ARNt Sintetasas/genética , Ciclodextrinas/química , ADN Polimerasa III/genética , Escherichia coli/enzimología , Escherichia coli/genética , Flúor/química , Interacciones Hidrofóbicas e Hidrofílicas , Isomerasas/metabolismo , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Propiedades de Superficie , Ubiquitina/químicaRESUMEN
Hydrophobins are a family of cysteine-rich proteins unique to filamentous fungi. The proteins are produced in a soluble form but self-assemble into organised amphipathic layers at hydrophilic:hydrophobic interfaces. These layers contribute to transitions between wet and dry environments, spore dispersal and attachment to surfaces for growth and infection. Hydrophobins are characterised by four disulphide bonds that are critical to their structure and function. Thus, obtaining correctly folded, soluble and functional hydrophobins directly from bacterial recombinant expression is challenging and in most cases, initial denaturation from inclusion bodies followed by oxidative refolding are required to obtain folded proteins. Here, we report the use of cell-free expression with E. coli cell lysate to directly obtain natively folded hydrophobins. All six of the hydrophobins tested could be expressed after optimisation of redox conditions. For some hydrophobins, the inclusion of the disulfide isomerase DsbC further enhanced expression levels. We are able to achieve a yield of up to 1 mg of natively folded hydrophobin per mL of reaction. This has allowed the confirmation of the correct folding of hydrophobins with the use of 15N-cysteine and 15N-1H nuclear magnetic resonance experiments within 24 h of starting from plasmid stocks.