Vitamin B12 and BtuF protein interaction analysis method

A protein interaction and analysis method technology, which is applied in the analysis field of vitamin B12 and BtuF protein interaction, can solve the problems of protein molecular weight limitation, time-consuming map analysis, large sample volume, etc., to achieve less sample volume and sample purity The effect of low cost and simple operation method

Active Publication Date: 2017-05-31
NANJING UNIV OF SCI & TECH
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Problems solved by technology

[0005] Aiming at the disadvantages of traditional protein structural biology research methods, such as low sensitivity, limitation on protein molecular weight, large amount of sample required, high purity of required sample and time-consuming analysis of graphs, etc., the present invention provides a vitamin B12 and BtuF protein Interaction analysis method can realize efficient and accurate detection and analysis of protein-ligand interaction

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  • Vitamin B12 and BtuF protein interaction analysis method
  • Vitamin B12 and BtuF protein interaction analysis method
  • Vitamin B12 and BtuF protein interaction analysis method

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Embodiment 1

[0024] In the first step, BtuF protein and all protein standards (including alcohol dehydrogenase, concanavalin, avidin, β-lactoglobulin, myosin, cytochrome c, and serum amyloid) were prepared in 10kD The ultrafiltration membrane was desalted, the centrifugation temperature was 4°C, the centrifugation speed was 14000rcf, and the centrifugation was 30min, repeated 5 times, wherein the desalted BtuF protein was replaced with pH8. into the ammonium acetate buffer at pH 7.4;

[0025] In the second step, an equimolar amount of vitamin B12 solution prepared with an ammonium acetate buffer solution of pH 8.0 is added to the BtuF protein solution obtained in the first step to obtain a BtuF-VB complex solution;

[0026] The third step is to draw 2 μL of BtuF protein and BtuF-VB with a final concentration of 10uM and a final concentration of 5uM protein standard for mass spectrometry, and adjust the instrument parameters such as: the working mode is ion mobility mode, the ionization mod...

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Abstract

The invention discloses a vitamin B12 and BtuF protein interaction analysis method. According to the method, BtuF protein is replaced into pH8.0 ammonium acetate buffer solution, vitamin B12 is added according to the mole ratio of 1:1 to prepare BtuF-VB solution, the BtuF protein, a BtuF-VB protein compound and a protein standard substance are subjected to mass spectrometric analysis under the same mass spectrum condition, ion mobility data of the BtuF protein, the BtuF-VB protein compound and the protein standard substance are acquired and processed to obtain respective drift time distribution, the collision cross section area of the BtuF and the BtuF-VB is acquired according to drift time distribution and the theoretical collision cross section area of the protein standard substance and drift time distribution of the BtuF protein and the BtuF-VB, BtuF-VB interaction is analyzed according to drift time distribution and the collision cross section area, and the vitamin B12 and the BtuF protein are better in structural uniformity and more compact in conformation after combination. The ion mobility mass spectrometry method is simple in operation, less in needed sample amount and good in repeatability, and can rapidly and accurately provide protein structure information.

Description

technical field [0001] The invention belongs to the technical field of protein structure biology, and in particular relates to an analysis method for the interaction between vitamin B12 and BtuF protein. Background technique [0002] The interaction and recognition between proteins and ligands is an important way for proteins to exert their biological functions, and plays a very important role in various life activities. Therefore, understanding the interaction mechanism between proteins and ligands is very important for understanding the regulation of organisms. Mechanisms are crucial. The vitamin B12 absorption system in Escherichia coli consists of BtuB, BtuF, and BtuCD. After BtuF captures VB12 in the periplasm, it docks with BtuCD to form a stable BtuCDF complex, and starts the transport of VB12 from the outside to the inside of the membrane. [0003] There is only one hinge-like "connector" between the two spherical domains of BtuF, which belongs to class III in the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/62
Inventor 周敏龚晓芸
Owner NANJING UNIV OF SCI & TECH
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