Nucleant-mediated protein crystallization with the application of microporous synthetic zeolites

Sugahara, Michihiro; Asada, Yujiro; Morikawa, Yuko; Kageyama, Yuichi; Kunishima, N.

doi:10.1107/s0907444908009980

Cited by 53 publications

(61 citation statements)

References 44 publications

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“…S4). Other structural homologs, as identified in an earlier study based on YTHDC1 (13), include the DUF55 domain of human thymocyte nuclear protein 1 (15) and the EVE domain that is found in a collection of prokaryotic proteins (16) such as Pyrococcus horikoshii protein PH1033 (17), Agrobacterium tumefaciens Atu2648 (16), and a Leishmania major protein (18) (Fig. S4), with Z scores between 9 and 13 from the program DaliLite (19) and sequence identities between 10 and 18%.…”

Section: Resultsmentioning

confidence: 99%

Molecular basis for the recognition of methylated adenines in RNA by the eukaryotic YTH domain

Luo

Tong

2014

Proc. Natl. Acad. Sci. U.S.A.

209

237

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Methylation of the N6 position of selected internal adenines (m 6 A) in mRNAs and noncoding RNAs is widespread in eukaryotes, and the YTH domain in a collection of proteins recognizes this modification. We report the crystal structure of the splicing factor YT521-B homology (YTH) domain of Zygosaccharomyces rouxii MRB1 in complex with a heptaribonucleotide with an m 6 A residue in the center. The m 6 A modification is recognized by an aromatic cage, being sandwiched between a Trp and Tyr residue and with the methyl group pointed toward another Trp residue. Mutations of YTH domain residues in the RNA binding site can abolish the formation of the complex, confirming the structural observations. These residues are conserved in the human YTH proteins that also bind m 6 A RNA, suggesting a conserved mode of recognition. Overall, our structural and biochemical studies have defined the molecular basis for how the YTH domain functions as a reader of methylated adenines.T he methylation of the N6 position of selected internal adenines (m 6 A) modification is widespread in eukaryotic mRNAs and noncoding RNAs (1-3), reviewed in refs. 4-6. Recent studies have linked this modification to the regulation of alternative splicing (2), RNA processing, and mRNA degradation (7). Although the exact cellular functions of this modification are still not completely understood, m 6 A has been linked to the regulation of the circadian clock (8) and m 6 A levels are highest during yeast meiosis (1). The m 6 A methyl group can be removed by the dioxygenases FTO (9) and ALKBH5 (10, 11), suggesting that m 6 A is a reversible modification on the RNA.A consensus sequence G(m 6 A)C has been identified for this modification based on transcriptome-wide mapping (1, 2), which is consistent with that identified from earlier biochemical studies (4-6). Several YTH domain (12) family members, YTHDF1, YTHDF2, and YTHDF3 in humans (2, 7) and methylated RNAbinding protein 1 (MRB1) in yeast (1), have been shown to bind RNAs with m 6 A modification, and the binding consensus for the YTH domain of YTHDF2 is also G(m 6 A)C (7), consistent with that found by transcriptome-wide mapping.The YTH domain contains ∼160 residues and is found in yeast, plants, and animals ( Fig. S1) (12, 13). The domain is located at the C-terminal end of yeast MRB1 and human YTHDF1-3 (Fig. 1A), and its sequence is well conserved among these proteins (Fig. 1B and Fig. S1). The N-terminal regions of these proteins are poorly conserved, although that of YTHDF2 mediates its function in regulating mRNA localization and degradation (7). Yeast MRB1 regulates phosphate metabolism by destabilizing the mRNA of a transcription factor of the pathway and, hence, it is also known as Pho92 (14), although direct evidence of MRB1 regulating m 6 A-containing mRNAs in yeast cells is lacking.The structures of the YTH domains of two related proteins, human YTH domain containing protein 1 [YTHDC1; Protein Data Bank (PDB) ID code 2YUD] and YTHDC2 (2YU6), have been reported. Human YTHDC1 has 29% sequence i...

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Section: Resultsmentioning

confidence: 99%

Molecular basis for the recognition of methylated adenines in RNA by the eukaryotic YTH domain

Luo

Tong

2014

Proc. Natl. Acad. Sci. U.S.A.

209

237

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“…A crystallization drop of 1.0 µL, in the presence of synthetic zeolite molecular sieves as heteroepitaxic nucleants [40], [41], was created by mixing 1∶1 mixture of 28.0 mg/mL protein solution in 0.02 M MES-NaOH (pH 5.8) and precipitant solution composed of 40% ( w/v ) polyethylene glycol (PEG) 8,000, 0.2 M CaCl 2 , and 0.1 M MES-NaOH (pH 5.8) in a well of a Nunc HLA crystallization plate (Nalge Nunc International) which was then covered with 20 µL of paraffin oil. The resulting Ao α-amylase crystals were submitted to a heavy-atom derivatization experiment.…”

Section: Methodsmentioning

confidence: 99%

Effect of Heavy Atoms on the Thermal Stability of α-Amylase from Aspergillus oryzae

Sugahara

Takehira

2013

PLoS ONE

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Currently, there are no versatile and established methods for improving stability of proteins. In an entirely different approach from conventional techniques such as mutagenesis, we attempted to enhance enzyme stability of α-amylase from Aspergillus oryzae using a heavy-atom derivatization technique. We evaluated changes in stability using differential scanning calorimetry (DSC). Candidate heavy atoms were identified using the Heavy-Atom Database System HATODAS, a Web-based tool designed to assist in heavy-atom derivatization of proteins for X-ray crystallography. The denaturation temperature of α-amylase derivatized with gadolinium (Gd) or samarium (Sm) ions increased by 6.2 or 5.7°C, respectively, compared to that of the native protein (60.6°C). The binding of six Gd ions was confirmed by X-ray crystallography of the enzyme at 1.5 Å resolution. DSC and dynamic light-scattering data revealed a correlation between stability and the aggregation state upon addition of Gd ions. These results show that HATODAS search is an effective tool for selecting heavy atoms for stabilization of this protein.

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“…The pores promote aggregation of proteins such that a spontaneous critical nucleus comprised of tens of protein molecules form inside each pore. Another study with respect to a porous material has shown that zeolites with regular micropores induce heteroepitactic nucleation [19]. A common feature of these nucleants is that their specific surface properties govern the nucleation potential of the proteins.…”

Section: Introductionmentioning

confidence: 99%

Heterogeneous Nucleation of Protein Crystals on Fluorinated Layered Silicate

et al. 2011

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Here, we describe an improved system for protein crystallization based on heterogeneous nucleation using fluorinated layered silicate. In addition, we also investigated the mechanism of nucleation on the silicate surface. Crystallization of lysozyme using silicates with different chemical compositions indicated that fluorosilicates promoted nucleation whereas the silicates without fluorine did not. The use of synthesized saponites for lysozyme crystallization confirmed that the substitution of hydroxyl groups contained in the lamellae structure for fluorine atoms is responsible for the nucleation-inducing property of the nucleant. Crystallization of twelve proteins with a wide range of pI values revealed that the nucleation promoting effect of the saponites tended to increase with increased substitution rate. Furthermore, the saponite with the highest fluorine content promoted nucleation in all the test proteins regardless of their overall net charge. Adsorption experiments of proteins on the saponites confirmed that the density of adsorbed molecules increased according to the substitution rate, thereby explaining the heterogeneous nucleation on the silicate surface.

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Nucleant-mediated protein crystallization with the application of microporous synthetic zeolites

Cited by 53 publications

References 44 publications

Molecular basis for the recognition of methylated adenines in RNA by the eukaryotic YTH domain

Molecular basis for the recognition of methylated adenines in RNA by the eukaryotic YTH domain

Effect of Heavy Atoms on the Thermal Stability of α-Amylase from Aspergillus oryzae

Heterogeneous Nucleation of Protein Crystals on Fluorinated Layered Silicate

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