Bioconjugation of <scp>l</scp>-3,4-Dihydroxyphenylalanine Containing Protein with a Polysaccharide

Ayyadurai, Niraikulam; Nadarajan, Sivakumar; Deepankumar, Kanagavel; Jang, Yoon Jung; Chitrapriya, Nataraj; Song, Eun Kee; Lee, Nahum; Kim, Seog K.; Kim, Byung‐Gee; Soundrarajan, Nagasundarapandian; Lee, Sun‐Gu; Joon, Hyung; Budiša, Nediljko; Yun, Hyungdon

doi:10.1021/bc2000066

Cited by 52 publications

(40 citation statements)

References 19 publications

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“…Indeed, the concept was successfully employed in some typical protein engineering studies based on canonical amino acid mediated substitution mutagenesis [19], [20], [21]. We also demonstrated that the concept could be applied to non-canonical amino acid mediated protein engineering approach [22], which permitted the generation of functional proteins with unnatural properties efficiently [23], [24].…”

Section: Introductionmentioning

confidence: 83%

Deletional Protein Engineering Based on Stable Fold

et al. 2012

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Diversification of protein sequence-structure space is a major concern in protein engineering. Deletion mutagenesis can generate a protein sequence-structure space different from substitution mutagenesis mediated space, but it has not been widely used in protein engineering compared to substitution mutagenesis, because it causes a relatively huge range of structural perturbations of target proteins which often inactivates the proteins. In this study, we demonstrate that, using green fluorescent protein (GFP) as a model system, the drawback of the deletional protein engineering can be overcome by employing the protein structure with high stability. The systematic dissection of N-terminal, C-terminal and internal sequences of GFPs with two different stabilities showed that GFP with high stability (s-GFP), was more tolerant to the elimination of amino acids compared to a GFP with normal stability (n-GFP). The deletion studies of s-GFP enabled us to achieve three interesting variants viz. s-DL4, s-N14, and s-C225, which could not been obtained from n-GFP. The deletion of 191–196 loop sequences led to the variant s-DL4 that was expressed predominantly as insoluble form but mostly active. The s-N14 and s-C225 are the variants without the amino acid residues involving secondary structures around N- and C-terminals of GFP fold respectively, exhibiting comparable biophysical properties of the n-GFP. Structural analysis of the variants through computational modeling study gave a few structural insights that can explain the spectral properties of the variants. Our study suggests that the protein sequence-structure space of deletion mutants can be more efficiently explored by employing the protein structure with higher stability.

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Section: Introductionmentioning

confidence: 83%

Deletional Protein Engineering Based on Stable Fold

et al. 2012

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“…Similarly, 3,4-dihydroxy-L-phenylalanine (L-DOPA, Fig. 2 (10)) can be oxidized by NaIO 4 to form a nucleophile-reactive orthoquinone intermediate [72].…”

Section: Bioorthogonal Labeling Of Proteinsmentioning

confidence: 99%

Biochemical analysis with the expanded genetic lexicon

2012

Anal Bioanal Chem

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The information used to build proteins is stored in the genetic material of every organism. In nature, ribosomes use 20 native amino acids to synthesize proteins in most circumstances. However, laboratory efforts to expand the genetic repertoire of living cells and organisms have successfully encoded more than 80 nonnative amino acids in E. coli, yeast, and other eukaryotic systems. The selectivity, fidelity, and site-specificity provided by the technology have enabled unprecedented flexibility in manipulating protein sequences and functions in cells. Various biophysical probes can be chemically conjugated or directly incorporated at specific residues in proteins, and corresponding analytical techniques can then be used to answer diverse biological questions. This review summarizes the methodology of genetic code expansion and its recent progress, and discusses the applications of commonly used analytical methods.

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“…Protein purification, quantification, and fluorescence analysis Purification and quantification of the recombinant proteins were performed as described by Ayyadurai et al (2011b). Fluorescence analysis was carried out with 2 lM protein by exciting at 470 nm, and the emission maximum was recorded using a spectrofluorimeter equipped with digital software.…”

Section: Chemicalsmentioning

confidence: 99%

Biosynthetic substitution of tyrosine in green fluorescent protein with its surrogate fluorotyrosine in Escherichia coli

et al. 2011

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Introduction of a fluorine moiety into green fluorescent protein offers an interesting novel spectral variant. The calculated binding energy of fluorotyrosine (F-Tyr) (-8.42 kcal/mol) for tyrosyl tRNA synthetase was moderately higher than that of tyrosine (Tyr) (-8.36 kcal/mol). This result directly correlated with the expression level of F-Tyr containing GFP (38 mg/l), which was comparably higher than that of the parent GFP expression level (34 mg/l). Finally, we generated a model structure for GFP to assess possible interaction in the chromophore of the protein structure, which plays an important role in determining the spectral and folding behaviors of the F-Tyr incorporated GFP variant.

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Bioconjugation of l-3,4-Dihydroxyphenylalanine Containing Protein with a Polysaccharide

Cited by 52 publications

References 19 publications

Deletional Protein Engineering Based on Stable Fold

Deletional Protein Engineering Based on Stable Fold

Biochemical analysis with the expanded genetic lexicon

Biosynthetic substitution of tyrosine in green fluorescent protein with its surrogate fluorotyrosine in Escherichia coli

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