Crystal Structure of the N-terminal Domain of Ryanodine Receptor from <em>Plutella xylostella</em>

Bidhan Chandra Nayak; Jie Wang; Lianyun Lin; Weiyi He; Minsheng You; Zhiguang Yuchi

doi:10.3791/58568

Kristallstruktur des N-terminale Domäne des Ryanodin-Rezeptoren von Plutella xylostella

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Biochemistry

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JoVE Journal Biochemistry

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

Kristallstruktur des N-terminale Domäne des Ryanodin-Rezeptoren von Plutella xylostella

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11:31 min

November 30, 2018

DOI: 10.3791/58568-v

Bidhan Chandra Nayak*¹, Jie Wang*¹, Lianyun Lin*^1,2,3,4, Weiyi He^2,3,4, Minsheng You^2,3,4, Zhiguang Yuchi^1,2

¹Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, Collaborative Innovation Center of Chemical Science and Engineering, School of Pharmaceutical Science and Technology,Tianjin University, ²State Key Laboratory of Ecological Pest Control for Fujian/Taiwan Crops and Institute of Applied Ecology,Fujian Agriculture and Forestry University, ³Joint International Research Laboratory of Ecological Pest Control,Ministry of Education, Fuzhou, ⁴Fujian-Taiwan Joint Centre for Ecological Control of Crop Pests,Fujian Agriculture and Forestry University

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Overview

This article details the protocols for protein expression, purification, crystallization, and structure determination of the N-terminal domain of the ryanodine receptor from the diamondback moth (Plutella xylostella). Understanding this structure aids in elucidating the molecular mechanisms of protein function and insecticide resistance.

Key Study Components

Area of Science

Neuroscience
Structural Biology
Insect Physiology

Background

Ryanodine receptors are crucial for calcium signaling in cells.
Understanding their structure can inform insecticide development.
Insecticide resistance is a growing concern in pest management.
X-ray crystallography is the gold standard for protein structure determination.

Purpose of Study

To solve the structure of the ryanodine receptor domain.
To understand the molecular mechanisms of channel gating.
To provide templates for species-specific insecticide design.

Methods Used

Amplification of DNA corresponding to the protein of interest using PCR.
Gel extraction of DNA post-PCR.
X-ray crystallography for structure determination.
Analysis of protein function and insecticide action.

Main Results

High-resolution structure of the ryanodine receptor domain was obtained.
Revealed mechanisms of channel gating.
Provided insights for structure-based drug design.
Identified challenges for new protein crystallographers.

Conclusions

The study enhances understanding of ryanodine receptor function.
It offers a framework for developing new insecticides.
Highlights the importance of interdisciplinary skills in protein crystallography.

Frequently Asked Questions

What is the significance of the ryanodine receptor?

The ryanodine receptor plays a key role in calcium signaling, which is essential for various cellular functions.

How does X-ray crystallography work?

X-ray crystallography involves diffracting X-rays through a crystal to determine the arrangement of atoms within the protein.

What challenges do new protein crystallographers face?

They often struggle with the need for proficiency in biochemistry, biophysics, and computational skills.

Why is understanding insecticide resistance important?

Understanding resistance mechanisms can lead to the development of more effective and targeted insecticides.

What methods are used to purify proteins?

Common methods include affinity chromatography, ion exchange, and size exclusion chromatography.

How can this research impact pest management?

By providing insights into the molecular mechanisms of insecticide action, it can lead to the design of more effective pest control strategies.

In diesem Artikel beschreiben wir die Protokolle der Proteinbestimmung Ausdruck, Reinigung, Kristallisation und Struktur der N-terminalen Domäne des Ryanodin-Rezeptoren von Diamondback Moth (Plutella Xylostella).

Die Lösung der Struktur der Ryanodin-Rezeptor-Domäne kann bei unserem Verständnis des molekularen Mechanismus der Proteinfunktion, insektizid-Wirkung und Der Entwicklung der Insektizidresistenz helfen. Diese Maßnahme impliziert die Verwendung der Röntgenkristallographie zur Strukturillustration, die als Goldstandard für die Bestimmung der Proteinstruktur bei nahezu atomarer Auflösung gilt. Diese hochauflösende Struktur der Insektenryanodin-Rezeptor-Domäne zeigt den Mechanismus der Kanalgating und stellt eine wichtige Vorlage für die Entwicklung von artspezifischen Insektiziden mit strukturbasierten Arzneimitteldesign-Ansätzen.

Im Allgemeinen werden Personen, die neu mit dieser Methode sind, Schwierigkeiten haben, weil angehende Proteinkristallographen in Biochemie, Biophysik, Informatik und Mathematik beherrschen müssen. Beginnen Sie mit der Verstärkung der DNA, die dem Protein von Interesse durch Polymerase-Kettenreaktion entspricht. Am Ende der Reaktion die gesamten 50 Mikroliter Reaktionsmischung auf einem 2%Agarose-Gel laufen lassen und die DNA mit einem Gelextraktionskit nach Standardprotokollen extrahieren.

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