Incorporation of non-canonicalaminoacids enables access to proteinsequencespace completely unknown to nature; the hope being that these new regions of sequencespace will contain improved protein stability or catalytic activity. Chemists have taken advantage of the properties fluorination provides polymers: thermal and oxidative stability, hydrophobicity, and inertness to chemical reactions. Protein engineers have utilized fluorinated aminoacids successfully in coiled-coiled protein interfaces lined by hydrophobic leucine residues, increasing melting temperatures up to 22 o C. Global incorporation of fluorinated aminoacids into globular proteins like chloramphenicol acetyltransferase has had the reverse effect, reducing enzyme melting temperature by up to 9 o C. Fluorinated aminoacids have also been limited by incorporation levels, creating proteins that are hybrids of natural and unnatural aminoacids. Quantitative incorporation of homoisoleucine, a larger aliphatic analogue of leucine, into chloramphenicol acetyltransferase results in decreased thermostability. Mutations that stabilized chloramphenicol acetyltransferase for 80%
stretch of thymidines. The 5’ sequence of the tRNA is removed by RNase P, and the 3’ end is removed by tRNase Z 24 . In mammalian cells, the 3’ CCA tail is added by a nucleotidyltransferase; 24 therefore, we tested tRNA sequences with and without a CCA tail (Figure S4.7-S4.9). We measured the activity of these various constructs for Anl incorporation by transiently transfecting several widely used mammalian cell lines, including Chinese hamster ovary (CHO) cells, monkey kidney cells (COS7) and an immortalized human cervical cancer cell line (HeLa). Cells were incubated in serum containing media supplemented with 1.5 mM Anl for a total of 10 hours and thereafter lysed. Total cellular proteins were treated with alkyne- tetramethylrhodamine (TAMRA) dye (Figure S4.10) for labeling via copper-catalyzed azide- alkyne cycloaddition (reaction 1, scheme 4.1b) 15,25,26 . After SDS-PAGE, TAMRA-labeled proteins were detected by in-gel fluorescence imaging (Figure 4.1d). As expected, Anl was not charged to any appreciable extent by the wild-type translation machinery of the cells. Anl incorporation was detected in cells carrying the E. coli NLL-MetRS. We did not observe a significant increase in Anl incorporation as a function of tRNA expression. The variability of incorporation across different cell lines suggests that the expression levels of the tRNA and the synthetase may need to be optimized for each cell type, thereby limiting the general applicability of this approach. Nonetheless, this strategy may prove to be useful for protein labeling in specific cases. For example, a similar strategy was used to incorporate several non-canonicalaminoacids using a genetically encoded pyrrolysyl-tRNA synthetase/tRNA pair in Drosophila. 27
4 th , 6 th , and 10 th were constructed in the laboratory and introduced into pAJL-61 plasmid, which also encodes for DHFR. DHFR synthesis was induced in the methionine-auxotroph M15MA, in methionine-free medium supplemented with 2.0 mM Anl. The SDS-PAGE analysis of cell lysates following DHFR expression is shown in Figure 3.5. The L13G mutant is known to allow close to quantitative replacement of Met sites with Anl in the presence of 1.0 mM Anl . This mutant is the only MetRS variant that supports DHFR synthesis at the conditions tested. DHFR production was not detected with other top ranking MetRS mutants. The appearance of high-ranking false positives may be due to the limited scope of the computational model. Some aspects of the biological system, such as binding site rearrangements linked to ligand binding, are not accounted for in the model and can lead to false positives. Alternatively, it is possible that the resolution of computational binding energies that are used to distinguish between mutants is lower than the resolution observed in experiments, such that two mutants that cannot be distinguished by binding energies might vary widely when their activities are determined in the laboratory. In order to better understand the limitations of the computational strategy on the MetRS system, additional mutation data was obtained through in vivo selection experiments. (Chapter 2)
Typically, directed evolution is used to create the novel tRNA and aminoacyl tRNA synthetase by encoding them on a plasmid. Then, they can be mutated either through error-prone PCR or the use of degenerate primers. Error-prone PCR uses a Taq polymerase that does not have a proof-reading capability, and the fidelity of the incorporation of nucleotides is controlled by altering the reaction buffer. Likewise, degenerate primers can be used to increase the mutation rate; they are a population of oligonucleotide sequences in which some positions can contain several possible bases. They cover all possible nucleotide sequence combinations for the targeted protein (Iserte et al. 2013). Following mutation, the library of tRNA/synthetase pairs can be examined via positive and negative selection to find those that display orthogonality. In the positive selection process, the plasmid is transferred to bacterial cells that contain genes that confer antibiotic resistance only in the presence of the ncAA. If a proper and functional orthogonal pair is expressed, the ncAA will be incorporated into an antibiotic resistance gene. The gene will be functional, and confer resistance to the antibiotic, allowing the bacteria to survive. In negative selection, the plasmid is transferred to cells that contain an essential gene that contains a premature amber stop codon. In the presence of the ncAA, the novel tRNA/synthetase pair successfully incorporates the ncAA, resulting in an essential, functional gene and the survival of the bacteria (Kato et al. 2015).
mutation was having multiple effects including increasing the cis bias of the P281 peptide bond was not patently excluded by the above analysis, so we attempted substitution of T280 with a variety of non-canonicalaminoacids to gain more insight into the role of this residue. It has been shown that the electron density of the aromatic ring adjacent to a proline correlates with increased proportions of the cis prolyl peptide bond, 20 so we attempted incorporation of a variety of 4-substituted phenylalanine analogs via non-canonicalamino acid mutagenesis. The aminoacids we attempted to incorporate are shown in Figure 4.12. Unfortunately, none of these non-canonicals incorporated well-enough in place of T280 in order for us to measure electrophysiological responses. We did, however, successfully incorporate O-methylthreonine (MeOThr) and the positive control, threonine, via the non- canonicalamino acid mutagenesis strategy. The resultant dose-response curves are shown in Figure 4.10B. Incorporation of the wild-type threonine gave responses very close to those of unmutated wild-type receptors, and incorporation of MeOThr led to a ~6-fold decrease in the EC 50 of 5-HT. Thu,s methylating the T280 side-chain hydroxyl group, both
this proline displays a distinctive phenotype: conventional mutants give a nonfunctional channel, but incorporation of α-hydroxy residues, regardless of side chain, yields robustly expressing, ligand-activated receptors. This establishes that of the several unique features of a proline residue, lack of a backbone hydrogen bond-donor is the essential requirement here. In GLIC, conventional mutants at Pro203 similarly gave negligible current responses. Surface expression of the P203L mutant was verified by western blotting of stripped oocyte membranes (data not shown), indicating that mutations at this site are capable of rendering the receptor nonfunctional, rather than interrupting assembly or trafficking. However, we were unable to rescue function by ablation of the backbone NH; α-hydroxy analogs gave nonfunctional channels. In fact, Pro203 proved to be extremely sensitive to substitution (Table 3.1; Figure 3.5D). Even the very close proline analogs Pip and Aze did not produce functional receptors. In contrast, cells injected with N-methylalanine (N-MeAla), which ablates the backbone NH but also lacks proline’s cyclic side chain, did yield large currents. The substituted proline analogs P3m and flp, as well as the unsaturated Dhp, also gave functional channels.
Genetic engineering provides a tool with which one can prepare complex macromolecules possessing both precisely controlled architectures and specific catalytic or biological activity. Recent work has shown the advantages of using the biosynthetic machinery to produce new materials (for a review see reference ). The use of monomers other than the twenty canonicalaminoacids enables the introduction of new functionality into proteins, creating the potential for novel physical and chemical properties. Analogues of many of the canonicalaminoacids have been incorporated into proteins in E. coli using the wild-type biosynthetic machinery, e.g. [2, 3], while modifications of that machinery have permitted the incorporation of a still broader set of non-canonicalaminoacids [4-14]. Increasing the number of amino acid monomers that can be incorporated into proteins, and thereby the range of physical properties and chemistries available, requires detailed understanding of the biosynthetic apparatus.
Figure 3.1 Mechanism of SNIPP - backbone cleavage induced by the photolysis of a protein containing a nitrophenylglycine (Npg) residue. .............................................................................................................69 Figure 3.2 Signaling is initiated by proteolysis of the extracellular N-terminal domain of protease- activated receptors. .....................................................................................................................................70 Figure 3.3 Schematic showing how site-specific backbone cleavage may disrupt the pathway of mechanical coupling between binding site conformational change and channel opening. .....................70 Figure 3.4 Presumed location of Cys loop in the nAChR, based on antibody binding studies and apparent glycosylation of Cys142. ............................................................................................................................72 Figure 3.5 Sequence of the Cys loop in all four subunits of embryonic mouse muscle nAChR..................74 Figure 3.6 Photolysis of nAChR containing Npg in non-alpha Cys loops leads to a consistent 50% reduction in whole-cell current. .................................................................................................................75 Figure 3.7 Whole-cell currents from oocytes exressing nAChR suppressed with Npg show both the significant background and clear effect of photolysis...............................................................................76 Figure 3.8 Western blot of total membrane preparations from oocytes expressing nAChR suppressed with Npg at position α 132, using Mab210 as the primary antibody. .......................................................78 Figure 3.9 Effect of the introduction of the HA epitope tag into nAChR subunits on whole-cell ACh current in response to 200 µ M ACh, 24 hr post-injection. .......................................................................79 Figure 3.10 Isolation of nAChR from the surface of Xenopus ooocytes by ultracentrifugation of homogenized oocytes followed by sub-cellular fractionation by sucrose step gradient..........................80 Figure 3.11 Assay for the plasma membrane-resident Na + /K + ATPase, showing that activity is greatest in
K11-linked chains are often accompanied by their K48 counterparts. Either of these homotypic conjugates can initiate protein degradation on their own , but their mutual incorporation, combined with chain branching, strongly enhances recognition by proteasomal receptor subunits in yeast [40, 47]. This may be in part ascribed to a conformation attained by the branched trimer, in which a novel hydrophobic interface between the distal Ub is found . In humans, K11/K48-branched chains assembled by UBR4 and UBR5 provide proteasome-dependent quality control of mitotic regulators, misfolded nascent polypeptides and pathological Huntingtin variants [48–50]. In Dros- ophila, the Ci family of transcription factors involved in Hedgehog signaling is modified with K11/K48-linked chains to control their activation and repression. Interestingly, this type of control can be achieved by either complete or partial degradation, but for a long time it was unclear how proteasomes designate ubiquitinated proteins for the specific type of degradation. Some light was shed with the discovery that Ter94, a homolog of CDC48/p97/VCP, and K11-linked ubiquitination regulate partial degradation in Dros- ophila . This finding is particularly important since the Hedgehog family of proteins plays an evolutionarily conserved role during the development of metazoans, and dis- ruption of Hedgehog signaling results in developmental disorders and cancers.
Chemoselective ligations refer to a limited set of reactions that exhibit the ability to modify a specific chemical moiety in the presence of a large number of competing functionalities [4, 6, 11]. Our ability to introduce unnatural aminoacids expands the number of selective chemistries that can be accessed for modification of biomolecules. Chapter 7 describes efforts directed towards development of Pd(0) cross coupling chemistry as a chemoselective chemistry [70-72]. Use of Pd(0) chemistry requires introduction of either an aryl halide, as phenylalanine analogs, or terminally unsaturated moieties, as either phenylalanine or methionine analogs [73- 75]. Through the use of a model system and two protein systems we demonstrate that Pd(0) couplings satisfy the requirements for chemoselective ligations. These
incubator chamber using Dulbecco’s modified Eagle’s medium with 4.5 g/L glucose, 2 mM L-glutamine and phenol red (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies), 5% penicillin/streptomycin (P/S, Life Technologies). Every 3 days, at approximately 80% confluency, cells were subcultured and seeded in a 6-well plate at a cell density of 8x10 3 cells / cm 2 (or 8x10 4 cells per well) for 24 h prior to insulin addition. Insulins or vehicle were added directly to the medium at 200 nM (10 μL of a 50 μM solution in vehicle PBS) and incubated for 10 min prior to PBS washes to remove excess medium. HEK293 cells were lysed on-plate using IP Lysis Buffer (ThermoFisher, Pierce) with 50 U/mL benzonase nuclease (Sigma-Aldrich) for 20 min at 4°C; lysates were precipitated using ice cold acetone and re-suspended in 8 M urea, 20 mM Tris, pH 10.0. The protein concentration in the lysate was quantified by the BCA assay (ThermoFisher, Pierce) according to the manufacturer’s protocol and normalized for even protein loading across lanes. Lysates were separated by SDS-PAGE (4-12% Novex Bis/Tris SDS-PAGE gels, Life Technologies) in duplicate and transferred to a nitrocellulose membrane (Hybond ECL, GE Healthcare) using a wet transfer system. The membrane was blocked at RT in 5% nonfat milk in Tris-buffer saline with 0.1% Tween 20 (TBS/Tween) and washed with TBS/Tween prior to blotting with antibodies.
The nutritional value of lupine seeds, both to hu- mans and animals, results mainly from the quantity and quality of the seed proteins. An analysis of the total protein content showed that it varied in the examined materials and was dependent both on the cultivar and place of cultivation. Experiments confirmed high protein content in the examined blue lupine seeds (between 28% and 41% for the cultivars grown in Przebędowo, and between 29% and 39% in Wiatrowo) (Table 2). The worst cultivar in terms of the protein content, regardless of the field on which the cultivar had grown, was Kalif (average total protein content 29.37 ± 1.14%), while the best cultivar was Boruta (average total protein content 37.43 ± 0.98%). An unexpected effect was observed: cultivars with the lowest and the highest protein content differed depending on the place of cultivation. Even when the applied cultivation conditions were the same, the place of cultivation influenced the protein content in the derived seeds. The lowest content of protein in Wiatrowo was found in cvs. Salsa and Bojar seeds, the highest – in cv. Karo. In Przebędowo, the highest and lowest content of protein was
been removed. The resulting sticky yellow solid was dissolved in methanol (150 mL) and acetone (47 mL, 0.643 mol) and para-toluene sulfonic acid (0.22g, 0.128 mol) was then added. The solution was stirred under reflux for 18 h. Excess reagents and solvents were removed in vacuo. Care was taken to remove the methanol as remaining traces of the solvent affect the final crystallization step, due to the high solubility of the product in this solvent. Trifluoroacetic acid (30 mL) was added carefully to the resulting orange solid at 0 ˚C. The reaction was left to stir until the solid was dissolved. Trifluoromethanesulfonic acid (2.83 mL, 0.032 mol) was then added slowly and allowed to stir for 10 min before methacryloyl chloride (25 mL, 0.256 mol) was added slowly. 32 The reaction mixture was then allowed to warm to room temperature and stirred overnight. Excess solvents and acids were removed by air flow and the resulting product thoroughly dried under vacuum with a sintered attachment to prevent loss of any solids. A sticky yellow solid resulted and to this, diethyl ether (300 mL) was added, resulting in a white precipitate which was collected, further washed with diethyl ether (~ 100 mL) and dried in vacuo to give the desired MacMillan functionalized monomer M 3.1 as an amorphous white solid (33.4 g, 87%). 1 H NMR (300 MHz, CD 3 OD): δ 1.51 (3H, s, -
All computational protein design programs attempt to solve what has been called the inverse protein folding problem: given some structural information (usually the protein backbone conformation) find the sequence with the lowest free energy for that structure . There have been significant advances toward this goal. The earliest attempts came from simply looking at the structures of proteins and the commonalities between them; common targets were helical bundle proteins (reviewed by De Grado et al.  and Richardson et al. ). Work progressed to improve the packing of designed proteins using algorithms to find compatible side chain conformations with simple secondary structures  and eventually known protein backbones serving as scaffolds  including the first fully automated full sequence design of a protein by Dahiyat and Mayo . There have been additional advances toward the de novo design of proteins with arbitrary shape and unseen topology such as the design of a novel α/β protein fold by Kuhlman et al. , the design of a 4-helix bundle by Summa et al. , and the design a β-sheet protein by Kraemer-Pecore et al. . Recently protein design has progressed to the point where the design of enzymes is possible [10, 11].
About a third of all proteins in nature are estimated to use metal ions to perform their function. 82, 83 Metals are important structural elements in many proteins, are critical for regulating a number of biological activities, and are key cofactors in enzyme catalysis and electron transfer processes. Proteins that bind to metal ions (metalloproteins) are involved in important biological processes such as photosynthesis, oxygen transport, nitrogen fixation, synthesis and degradation of biomolecules, and water oxidation. 83, 84 Due to the critical roles that are played by metalloproteins, considerable efforts have been devoted to understanding their structures and functions. Moreover, there is an interest in designing metalloproteins with new or enhanced functions, catalytic activities, and structural characteristics, in order to manipulate biological processes. 85, 86 However, the design of artificial metalloproteins using the common 20 aminoacids is limited, as the precise orientation of multiple amino acid side- chains is often required to create a defined metal ion binding site. This is crucial as localization of the metal complex is critical for the protein’s structure and function.
Abstract : Gas chromatography–mass spectrometry (GC-MS) is a widely used analytical technique in metabolomics. GC provides the highest resolution of any standard chromatographic separation method, and with modern instrumentation, retention times are very consistent between analyses. Electron impact ionization and fragmentation is generally reproducible between instruments and extensive libraries of spectra are available that enhance the identification of analytes. The major limitation is the restriction to volatile analytes, and hence the requirement to convert many metabolites to volatile derivatives through chemical derivatization. Here we compared the analytical performance of two derivatization techniques, silylation (TMS) and alkylation (MCF), used for the analysis of amino and non-amino organic acids as well as nucleotides in microbial-derived samples. The widely used TMS derivatization method showed poorer reproducibility and instability during chromatographic runs while the MCF derivatives presented better analytical performance. Therefore, alkylation (MCF) derivatization seems to be preferable for the analysis of polyfunctional amines, nucleotides and organic acids in microbial metabolomics studies.
Data Collection and Statistical Analyses. A randomized complete block design was applied with random allocation of dietary treatments to two pens within each brooding room in the experimental facility, to provide eight replicate pens per treatment. Group body weights were measured at 1 d and 14 d, with individual bird BW in each pen recorded at 20 d. Mortality was collected twice daily and weighed to calculate the AdjFCR of pens at weekly intervals to 20 d of age. At 20 d, two pens per treatment were weighed to derive an estimate of the mean BW. Subsequently, individual BW of all birds were recorded and all birds with a BW falling in the range of + 20 g of the mean were marked with black paint. Feed was withdrawn 10 h before processing, while allowing continued access to water. At 21 d, two marked birds per pen were chosen at random for processing. The processing sequence consisted of bleeding, scalding, plucking and evisceration with care being taken not to remove the fat pad during the manual evisceration. Subsequently the dry carcass weight without shanks, feet, and neck was recorded prior to removal of the fat pad. Carcasses were then further processed into pre-defined cuts consisting of back half, wings, breast skin, and Pectoralis major and Pectoralis minor breast muscles.
What happens to a photon traveling through an empty universe? A simple (and correct) answer is that nothing. Quoting the lyrics of a song by Grzegorz Turnau, “in fact, nothing happens and nothing occurs till the very end”. The end occurs when the emitted photon is finally absorbed by an observer’s eye, a photographic plate, or a CCD device. This absorption reveals that the frequency of the photon is redshifted. The only possible interpretation of this redshift is a Doppler shift, due to relative motion of the emitter and the absorber. In an empty universe, these motions can be described entirely by means of the Milne kinematic model. In this model, the cosmic arena of physical events is the pre-existing Minkowski spacetime. In the origin of the coordinate system, O, at time t = 0 an ‘explosion’ takes place, sending radially Fundamental Observers (FOs) with constant velocities in the range of speeds (0, c). Let’s place a source of radiation at the origin of the coordinate system. At time t e the source emits