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•NT* fusion of human rhinovirus 14 3C protease for high-yield expression.•Stable protease for tag removal at low temperature in minutes.•Alternative to tag cleavage with TEV protease.
...The human rhinovirus 14 3C protease (HRV3C protease), in fusion with glutathione S-transferase also referred to as PreScission™ protease, is a cysteine protease of particular interest for affinity tag removal from fusion proteins due to its stringent recognition sequence specificity (LEVLFQ/GX) and superior activity at low temperature. Here we report the expression, purification and use of a fusion construct of HRV3C protease, NT*-HRV3CP, that affords high expression yield in E. coli (over 300 mg/L cell culture), facile single-step purification, high solubility (>10 mg/mL) and excellent storage properties. NT*-HRV3CP cleaves affinity tags at 4 °C in minutes, making it an attractive tool for the production of recombinant proteins for biotechnological, industrial and pharmaceutical applications.
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.
Global substitution of leucine for analogues containing CH
F instead of methyl groups delivers proteins with multiple sites for monitoring by
F nuclear magnetic resonance (NMR) spectroscopy. The 19 ...kDa
peptidyl-prolyl
isomerase B (PpiB) was prepared with uniform high-level substitution of leucine by (2
,4
)-5-fluoroleucine, (2
,4
)-5-fluoroleucine, or 5,5'-difluoroleucine. The stability of the samples toward thermal denaturation was little altered compared to the wild-type protein.
F nuclear magnetic resonance (NMR) spectra showed large chemical shift dispersions between 6 and 17 ppm. The
F chemical shifts correlate with the three-bond
H-
F couplings (
), providing the first experimental verification of the γ-gauche effect predicted by Feeney, J.
1996, 118, 8700-8706 and establishing the effect as the predominant determinant of the
F chemical shifts of CH
F groups. Individual CH
F groups can be confined to single rotameric states by the protein environment, but most CH
F 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 CH
F rotamers in agreement with results obtained previously for 1,3-difluoropropane. The sensitivity of the
F chemical shift to the rotameric state of the CH
F groups potentially renders them particularly sensitive for detecting allosteric effects.
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 (3 J HF), 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.
Efficient syntheses of fluorinated leucines, valines and alanines are described. The synthetic routes provide expedient access to various
C/
N/D isotopologues requiring solely readily available and ...inexpensive isotope containing reagents such as NaBD
, carbon-
C dioxide and sodium azide-1-
N. 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
, could be incorporated into proteins this way.
F-NMR spectra of the protein GB1 produced with these amino acids showed large chemical shift dispersions. Particularly high incorporation yields and clean
F-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.
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 N 6-(((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.
It is an open question whether the conformations of proteins sampled in dilute solutions are the same as in the cellular environment. Here we address this question by double electron-electron ...resonance (DEER) distance measurements with Gd(III) spin labels to probe the conformations of calmodulin (CaM) in vitro, in cell extract, and in human HeLa cells. Using the CaM mutants N53C/T110C and T34C/T117C labeled with maleimide-DOTA-Gd(III) in the N- and C-terminal domains, we observed broad and varied interdomain distance distributions. The in vitro distance distributions of apo-CaM and holo-CaM in the presence and absence of the IQ target peptide can be described by combinations of closed, open, and collapsed conformations. In cell extract, apo- and holo-CaM bind to target proteins in a similar way as apo- and holo-CaM bind to IQ peptide in vitro. In HeLa cells, however, in the presence or absence of elevated in-cell Ca
levels CaM unexpectedly produced more open conformations and very broad distance distributions indicative of many different interactions with in-cell components. These results show-case the importance of in-cell analyses of protein structures.
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) in‐cell macrocyclization of peptides and proteins, and iii) 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.
Genetic encoded cyanopyridylalanines are presented as convenient and highly selective reactive groups for in vivo functionalization, cyclization and stapling of polypeptides. The non‐toxic compounds can be genetically incorporated into proteins with high yields and their biocompatible reaction with amino‐thiol moieties proceeds under physiological conditions to near completion without requiring a catalyst.
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.
Calmodulin (CaM) is a calcium-binding protein that regulates the function of many proteins by indirectly conferring Ca2+ sensitivity, and it undergoes a large conformational change on partners' ...binding. We compared the solution binding mode of the target peptides MARCKS and IQ by double electron-electron resonance (DEER) distance measurements and paramagnetic NMR. We combined nitroxide and Gd(III) spin labels, including specific substitution of one of the Ca2+ ions in the CaM mutant N60D by a Gd(III) ion. The binding of MARCKS to holo-CaM resulted neither in a closed conformation nor in a unique relative orientation between the two CaM domains, in contrast with the crystal structure. Binding of IQ to holo-CaM did generate a closed conformation. Using elastic network modeling and 12 distance restraints obtained from multiple holo-CaM/IQ DEER data, we derived a model of the solution structure, which is in reasonable agreement with the crystal structure.
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•The target peptides MARCKS and IQ bind CaM both in its apo- and holo-states•In solution, the binding of MARCKS to holo-CAM does not generate a closed conformation•In solution, holo-CaM with bound IQ has a closed conformation, albeit heterogeneous•Gd(III)/nitroxide DEER distance restraints give a solution holo-CaM/IQ structural model
Jash et al. track the conformational changes calmodulin undergoes on binding MARCKS and IQ peptides in solution and report significant differences in their binding modes. This is achieved by various Gd(III) and nitroxide spin labeling schemes for CaM and the peptides combined with EPR inter-spin distance and paramagnetic NMR measurements.
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