The N‐terminal fragment of <i>Acanthamoeba polyphaga</i> mimivirus tyrosyl‐tRNA synthetase (TyrRS<sub>apm</sub>) is a monomer in solution

Choudhury, Aparajita; Banerjee, Rajat

doi:10.1016/j.febslet.2013.01.048

Cited by 3 publications

(4 citation statements)

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“…Sedimentation equilibrium experiments were performed in a Beckmann XL1-optima ultracentrifuge at 25 C (Beckman-Coulter Instruments) using An50-Ti rotor as reported previously 22 . Briefly 110 ll of each protein sample was loaded against 120 ll buffer solutions in a 12 mm charcoal filled six-chambered EPON centerpiece.…”

Section: Analytical Ultracentrifugation Studymentioning

confidence: 99%

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Spectroscopic studies of the unfolding of a multimeric protein α‐crystallin

et al. 2014

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α-Crystallin is a multimeric eye lens protein having molecular chaperone-like function which is crucial for lens transparency. The stability and unfolding-refolding properties of α-crystallin plays important roles for its function. We undertook a multi probe based fluorescence spectroscopic approach to explore the changes in the various levels of organization of this protein at different urea concentration. Steady state fluorescence studies reveal that at 0.6M urea a compact structural intermediate is formed which has a native-like secondary structure with enhanced surface exposure of hydrophobic groups. At 2.8M urea the tertiary interactions are largely collapsed with partial collapse of secondary and quaternary structure. The surface solvation probed by picosecond time resolved fluorescence of acrylodan labeled α-crystallin revealed dry native-like core of α-crystallin at 0.6M urea compared to enhanced water penetration at 2.8M urea and extensive solvation at 6M urea. Activation energy for the subunit exchange decreased by 22 kJ mol(-1) on changing urea concentration from 0 to 0.6M compared with over 75 kJ mol(-1) on changing urea concentration from 0 to 2.8M. Light scattering and analytical ultracentrifugation techniques were used to determine size and oligomerization of the unfolding intermediates. The data indicated swelling but no oligomer breakdown at 0.6M urea. At 2.8M urea the oligomeric size is considerably reduced and a monomer is produced at 6M urea. The data clearly reveals that structural breakdown of α-crystallin does not follow hierarchical sequence as tertiary structure dissolution takes place before complete oligomeric dissociation.

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Section: Analytical Ultracentrifugation Studymentioning

confidence: 99%

“…Partial specific volume was calculated as reported previously. 12,22 Data were fitted to a monomer n-mer self-association model where n was allowed to vary in Sedphat software. Quality of the fits was assessed by rmsd values.…”

Section: Analytical Ultracentrifugation Studymentioning

confidence: 99%

Spectroscopic studies of the unfolding of a multimeric protein α‐crystallin

et al. 2014

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“…However, in the previous studies we have reported that the isolated N-terminal containing entire CP1 domain, responsible for the dimerization of TyrRSapm, exists as monomer in solution. We argued that the C-terminal anticodon binding domain of TyrRSapm might have an indirect role in dimer formation [5]. The non-canonical dimeric interface organization due to unusual CP1 domain [2], indirect role of the C-terminal domain in non-canonical dimer formation [5] and speculated probable "4th domain of life" [6], all these idiosyncratic features evoked the question whether the noncanonical dimer interface has any implication on the structural and functional organization of TyrRSapm.…”

Section: Introductionmentioning

confidence: 98%

Unusual Role of Tryptophan residues in Structural and Functional properties of Mimi Virus TyrRS (TyrRS_apm)

Choudhury

Banerjee

2019

International Journal of Chemical and Environmental Sciences

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In crystal structure, the homodimeric (α2) mimi-virus Tyrosyl-tRNA Synthetases (TyrRSapm) showed significant variation in dimer orientation as compared to other solved structures of TyrRSs. Previous report showed that Bacillus stearothermophilus N-terminal TyrRS exists as dimer under native condition and unfolds through a monomeric intermediate. In our previous studies, we have reported that the C-terminal anticodon binding domain of TyrRSapm might have an indirect role in noncanonical dimer formation. To study the implication of the noncanonical dimer interface on the structural and functional organization of TyrRSapm, we replaced all three W residues (both of dimer interface and C-terminal domain) systematically with F residues (using site directed mutagenesis) with four different combinations (W80F/W120F, W278, W80F/W278F, W120F/W278F). We took advantage of Trp (W) fluorescence as the biophysical reporter for probing dimer interface study. The circular dichroism (CD) spectra of Trp mutants are dramatically different from wild-type indicating a huge conformation reorientation due to mutations. Another unusual feature of this enzyme is that it contains 10 cysteine residues per monomer and none of them involved in disulphide bridge formation. Using DTNB reaction as a probe again it was observed that mutants behave differently indicating a major perturbation. Fluorescence anisotropy supported by analytical ultracentrifugation data showed that TyrRSapm and its mutants exist as dimer. The unfolding pattern of N-terminal and C-terminal domain shows significant difference from each other indicating differential melting of domains. Aminoacylation reaction showed that two of the four Trp. mutants retain enzyme activities though different as compared to WT, while the other two mutants lost their activities. Hence it can be concluded that, the Trp residues play a crucial role in the structural as well as functional organization of TyrRSapm that may be reflected as the non canonical orientation of dimer conformation in crystal structure.

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“…Aminoacyl-tRNA synthetases are thought to have been central to the origin of life, participating in the transition from the RNA world to the realm of proteins (Brown and Doolittle 1995;Choudhury and Banerjee 2013;Nagel and Doolittle1995;Ribas de Pouplana and Schimmel 2001;Woese et al 2000). Increasingly, these conserved, universal enzymes have been identified as participants in more recently evolved and divergent cellular processes (Martinis et al 1999;Francklyn et al 2002).…”

Section: Introductionmentioning

confidence: 99%

VEGF, not VEGFR2, is associated with the angiogenesis effect of mini-TyrRS/mini-TrpRS in human umbilical vein endothelial cells in hypoxia

et al. 2013

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The purpose of this study was to determine the relationship between VEGF and mini-TyrRS/mini-TrpRS in angiogenesis in hypoxic culture and to begin to comprehend their mechanism in angiogenesis. We designed a VEGF gene silencing assay by using lentivirus vectors, and then western blotting was used to determine the protein expression of VEGF, VEGFR2 and pVEGFR2 in three groups in hypoxic culture at 3, 6, 12, or 24 h: (1) untransfected human umbilical vein endothelial cells (HUVECs) (Control); (2) pGCSIL-GFP lentivirus vector-transduced HUVECs (Mock); and (3) pGCSIL-shVEGF lentivirus vector-transduced HUVECs (Experimental). We also detected the effects of mini-TyrRS/mini-TrpRS peptides on HUVEC proliferation, migration and tube formation after lentivirus vector transfection and VEGFR2 antibody injection. The results indicated that expression of the mini-TyrRS protein was increased, whereas that of mini-TrpRS was specifically decreased in hypoxic culture both in control and mock groups. However, this trend in protein levels of mini-TyrRS and mini-TrpRS was lost in the experimental group after transduction with the pGCSIL-shVEGF lentivirus vector. The protein expression of VEGF was increased in hypoxic culture both in control and mock groups. After transduction with the pGCSIL-shVEGF lentivirus vector, the protein level of VEGF was noticeably decreased in the experimental group; however, for VEGFR2, the results showed no significant difference in VEGFR2 protein expression in any of the groups. For pVEGFR2, we found a distinct trend from that seen with VEGF. The protein expression of pVEGFR2 was sharply increased in hypoxic culture in the three groups. The addition of mini-TyrRS significantly promoted proliferation, migration and tube formation of HUVECs, while mini-TrpRS inhibited these processes in both control and mock groups in hypoxic culture. However, these effects disappeared after transduction with the pGCSIL-shVEGF lentivirus vector in the experimental group, but no significant difference was observed after VEGFR2 antibody injection. The protein expression of VEGF is similar to that of mini-TyrRS in hypoxic culture and plays an important role in the mini-TyrRS/mini-TrpRS-stimulated proliferation, migration and tube formation of HUVECs in hypoxia. These results also suggest that the change in mini-TyrRS and mini-TrpRS expression in hypoxic culture is not related to VEGFR2 and that some other possible mechanisms, are involved in the phosphorylation of VEGFR2.

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The N‐terminal fragment of Acanthamoeba polyphaga mimivirus tyrosyl‐tRNA synthetase (TyrRS_apm) is a monomer in solution

Cited by 3 publications

References 54 publications

Spectroscopic studies of the unfolding of a multimeric protein α‐crystallin

Spectroscopic studies of the unfolding of a multimeric protein α‐crystallin

Unusual Role of Tryptophan residues in Structural and Functional properties of Mimi Virus TyrRS (TyrRS_apm)

VEGF, not VEGFR2, is associated with the angiogenesis effect of mini-TyrRS/mini-TrpRS in human umbilical vein endothelial cells in hypoxia

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The N‐terminal fragment of Acanthamoeba polyphaga mimivirus tyrosyl‐tRNA synthetase (TyrRSapm) is a monomer in solution

Cited by 3 publications

References 54 publications

Spectroscopic studies of the unfolding of a multimeric protein α‐crystallin

Spectroscopic studies of the unfolding of a multimeric protein α‐crystallin

Unusual Role of Tryptophan residues in Structural and Functional properties of Mimi Virus TyrRS (TyrRSapm)

VEGF, not VEGFR2, is associated with the angiogenesis effect of mini-TyrRS/mini-TrpRS in human umbilical vein endothelial cells in hypoxia

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The N‐terminal fragment of Acanthamoeba polyphaga mimivirus tyrosyl‐tRNA synthetase (TyrRS_apm) is a monomer in solution

Unusual Role of Tryptophan residues in Structural and Functional properties of Mimi Virus TyrRS (TyrRS_apm)