• 한국자기공명학회논문지
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• 제21권2호
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• pp.50-54
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• 2017

Cupin-superfamily proteins represent the most functionally diverse groups of proteins and include a huge number of functionally uncharacterized proteins. Recently, YaiE, a cupin protein from Escherichia coli has been suggested to be involved in a novel activity of pyrimidine/purine nucleoside phosphorylase (PPNP). In the present study, we achieved a complete backbone NMR assignments of YaiE, by a series of heteronuclear multidimensional NMR experiments on its [$^{13}C/^{15}N$]-enriched sample. Subsequently, secondary structure analysis using the assigned chemical shift values identified 10 obvious ${\beta}-strands$ and a tentative $3_{10}-helix$. Taken all together, the results constitute the first structural characterization of a putative PPNP cupin protein.

• The Plant Pathology Journal
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• 제37권6호
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• pp.555-565
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• 2021

Bacteriophages infecting Acidovorax citrulli, the causal agent of bacterial fruit blotch, have been proven to be effective for the prevention and control of this disease. However, the occurrence of bacteriophage-resistant bacteria is one of hurdles in phage biocontrol and the understanding of phage resistance in this bacterium is an essential step. In this study, we aim to investigate possible phage resistance of A. citrulli and relationship between phage resistance and pathogenicity, and to isolate and characterize the genes involved in these phenomena. A phage-resistant and less-virulent mutant named as AC-17-G1 was isolated among 3,264 A. citrulli Tn5 mutants through serial spot assays and plaque assays followed by pathogenicity test using seed coating method. The mutant has the integrated Tn5 in the middle of a cupin protein gene. This mutant recovered its pathogenicity and phage sensitivity by complementation with corresponding wild-type gene. Site-directed mutation of this gene from wild-type by CRISPR/Cas9 system resulted in the loss of pathogenicity and acquisition of phage resistance. The growth of AC-17-G1 in King's B medium was much less than the wild-type, but the growth turned into normal in the medium supplemented with D-mannose 6-phosphate or D-fructose 6-phosphate indicating the cupin protein functions as a phosphomannos isomerase. Sodium dodecyl sulfa analysis of lipopolysaccharide (LPS) extracted from the mutant was smaller than that from wild-type. All these data suggest that the cupin protein is a phosphomannos isomerase involved in LPS synthesis, and LPS is an important determinant of pathogenicity and phage susceptibility of A. citrulli.

• BMB Reports
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• 제42권6호
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• pp.387-392
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• 2009

Here, we report the first biochemical and structural characterization of the hypothetical protein HP0902 from Helicobacter pylori, in terms of structural genomics. Gel-permeation chromatography and dynamic light scattering indicated that the protein behaves as a dimer in solution. Circular dichroism spectroscopy showed that HP0902 primarily adopts a $\beta$-structure and the protein was highly thermostable with a denaturing temperature higher than $70^{\circ}C$. Finally, the backbone NMR assignments were obtained on the [$^{13}C,^{15}N$]HP0902 and the secondary structure was determined using the chemical shift data. Additionally, the local flexibility was assessed via a heteronuclear $^1H-^{15}N$ steady state NOE experiment. The results revealed that HP0902 would adopt a compactly folded, all-$\beta$ topology with 11 $\beta$-strands. All of the results clearly support the notion that HP0902 belongs to the cupin superfamily of proteins.

• 한국환경농학회지
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• 제32권1호
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• pp.48-54
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• 2013

수수 종자의 품종 간 특이적으로 발현하는 단백질을 동정하여 기능성 유전자를 확보하고 이들 유전자를 이용하여 수수의 기능성 강화 및 품종 판별 기술 개발을 위한 유용 유전자를 확보하고자 프로테오믹스 기법을 이용하여 수수 종자로부터 단백질을 추출하였다. 추출한 단백질을 이차원전기영동후, colloidal CBB 염색을 통해 품종 별로 발현에 차이를 보이는 단백질을 분석하였다. 총 652 개의 spot들 중에 8개의 단백질 spot들이 발현 정도에 변화를 보였으며, 이들 단백질을 MALDI-TOF/TOF MS와 MASCOT database를 통해 동정한 결과, RNA metabolism (spot 1, spot 4) HSP (spot 2), 저장 단백질 (spot 3, spot 5, spot 6), 산화-환원 (spot 8) 관련 단백질 등이 동정되었다. 특히 동정된 단백질은 주로 흰찰수수 (WCS)에서 발현 정도가 높게 나오는 경향을 보였으며, 흰찰수수 (WCS)에서 유일하게 발현 되는 단백질로 Cupin family protein, Gloubulin 등이 동정되었다. DEAD-box helicase는 흰찰수수 (WCS)를 제외한 나머지 세 품종에서 발현되었다. Ribonuclease T2와 Aldo-Keto reductase는 대풍수수 (DPS)를 제외한 나머지 세 품종에서 발현되었다. HSPs는 토종수수 (TJS)에서만 발현 되는 것을 확인하였다. 이들 동정된 단백질들은 수수의 품종 별 특성을 이해하는데 중요한 단서를 제공할 것으로 예측된다.