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JoVE Journal

Biochemistry

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X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

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11:27 min

May 13th, 2020

May 13th, 2020

3,804 Views

0:04

Introduction

1:03

Activity Assay

2:19

Apoenzyme Preparation

3:32

Dimer Preparation

4:03

Microseeding

5:01

X-Ray Diffraction

5:48

Indexation

6:50

Molecular Replacement

8:04

Model Building

9:07

Results: Representative Thermotoga Maritima M42 Aminopeptidase TmPep1050 Oligomeric State Transition

10:50

Conclusion

Transcrição

This protocol facilitates the study of aminopeptidases with different oligomeric states and oligomerization-dependent activities that are balanced between inactive dimers and active dodecamers. This conventional technique can be easily performed i

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This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050H60A H307A variant.

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