Automatic Protein Crystallization in an Anaerobic Environment

Bret Dillard

B.C. Wang Lab

University of Georgia

07/21/06

Basis for Anaerobic Crystallization

Many proteins exist in an anaerobic environment

Lose cofactors such as iron-sulfur clusters in oxygen environment

Need to reproduce redox state of cofactors such as FAD⁺ and NAD⁺

Many crystallographers need to see different redox states of protein conformations for biological studies

Many Ways to Solve This Problem

Anaerobic experiments (in a chamber)

Sitting drop experiments

Hanging drop experiments

Capillary diffusion experiments

Or use Douglas robot in Bactron X chamber

Differences Between Techniques

Amount of Protein

Hanging drop, sitting drop, and capillary techniques use copious amounts of protein and crystallizing agent

Microbatch diffusion uses 300nl of protein per condition (in this experiment)

Microbatch allows use of same mother liquor for many experiments

The Chamber

Using Sheldon Manufacturing Bactron X chamber with custom wall joint one can run robot with computer outside the chamber

Connections and Oxygen Detection

24 parallel ports allow computer to run robot from outside the chamber

Using 5% hydrogen / 95% nitrogen gas mix we are allowed to detect oxygen levels inside the chamber

The same gas mix also allows us to use palladium catalyst to remove oxygen at an accelerated rate

The Overall Setup

Crystallization of Oxygen Sensitive Protein

We have purified and crystallized four proteins that are not stable in an oxygen environment

The structure of one of these proteins (rubrerythrin) has been solved using anaerobic techniques as of this moment.

This protein was previously solved with zinc being coordinated in an aerobic environment

The native protein contains iron but is unstable in oxygen

We now have the native iron form of this oxygen labile protein through using this system

Iron Form of Rubrerythrin

Advantages of Method

Use of less protein

Sitting drop, hanging drop, and capillary methods use large volumes of protein

Microbatch uses only 300nl of protein per condition

Less time involved in experiment

Sitting drop, hanging drop, and capillary methods require intensive attention per experiment

Microbatch requires only initial setup and the robot performs the experiment


	Many proteins exist in an anaerobic environment
		Lose cofactors such as iron-sulfur clusters in oxygen environment
		Need to reproduce redox state of cofactors such as FAD⁺ and NAD⁺
	Many crystallographers need to see different redox states of protein conformations for biological studies


	Anaerobic experiments (in a chamber)
		Sitting drop experiments
		Hanging drop experiments
		Capillary diffusion experiments
	Or use Douglas robot in Bactron X chamber


	Amount of Protein
		Hanging drop, sitting drop, and capillary techniques use copious amounts of protein and crystallizing agent
		Microbatch diffusion uses 300nl of protein per condition (in this experiment)
		Microbatch allows use of same mother liquor for many experiments


	Using Sheldon Manufacturing Bactron X chamber with custom wall joint one can run robot with computer outside the chamber


	24 parallel ports allow computer to run robot from outside the chamber
	Using 5% hydrogen / 95% nitrogen gas mix we are allowed to detect oxygen levels inside the chamber
	The same gas mix also allows us to use palladium catalyst to remove oxygen at an accelerated rate


	We have purified and crystallized four proteins that are not stable in an oxygen environment
	The structure of one of these proteins (rubrerythrin) has been solved using anaerobic techniques as of this moment.
		This protein was previously solved with zinc being coordinated in an aerobic environment
		The native protein contains iron but is unstable in oxygen
		We now have the native iron form of this oxygen labile protein through using this system


	Use of less protein
		Sitting drop, hanging drop, and capillary methods use large volumes of protein
		Microbatch uses only 300nl of protein per condition
	Less time involved in experiment
		Sitting drop, hanging drop, and capillary methods require intensive attention per experiment
		Microbatch requires only initial setup and the robot performs the experiment