High-throughput crystallographic screening device and method for crystalizing membrane proteins using a sub physiological resting membrane potential across a lipid matrix of variable composition

Patent metadata

Field	Value
Publication number	US-12128399-B2
Application number	US-202318225981-A
Country	US
Kind code	B2
Filing date	Jul 25, 2023
Priority date	Jun 29, 2016
Publication date	Oct 29, 2024
Grant date	Oct 29, 2024

Abstract

The invention is a high-throughput voltage screening crystallographic device and methodology that uses multiple micro wells and electric circuits capable of assaying different crystallization condition for the same or different proteins of interest at the same of different voltages under a humidity and temperature controlled environment. The protein is solubilized in a lipid matrix similar to the lipid composition of the protein in the native environment to ensure stability of the protein during crystallization. The invention provides a system and method where the protein is transferred to a lipid matrix that holds a resting membrane potential, which reduces the degree of conformational freedom of the protein. The invention overcomes the majority of the difficulties associated with vapor diffusion techniques and essentially reconstitutes the protein in its native lipid environment under “cuasi” physiological conditions.

First claim

What is claimed is:

1. A method of crystallizing one or more membrane proteins, said method comprising the step of incubating a membrane protein sample in a system, wherein the system comprises:
a sample unit comprising:
a sample holding layer having a plurality of holding wells, wherein each holding well comprises a pair of electrodes, and
a lid layer provided over said plurality of holding wells to seal the contents of said plurality of holding wells from each other; and

a variable direct current potential source electrically coupled to the pair of electrodes of each holding well, wherein the incubation of the membrane protein sample provides crystallization of the one or more membrane proteins.

2. The method of claim 1 , wherein the system further comprises:
an incubator receiving the sample unit;
a temperature unit monitoring and controlling a temperature inside the incubator; and
a humidity unit monitoring and controlling a humidity inside said incubator.

3. The method of claim 1 , wherein the variable direct current potential source is configured to alter a direction of a potential field generated between the pair of electrodes of each holding well, wherein the direction of the potential field generated varies within 180 degrees.

4. The method of claim 1 , wherein the plurality of holding wells has the same geometric shape or different geometric shapes.

5. The method of claim 1 , wherein said lid layer comprises a plurality of lid wells having the same shape and dimensions as said holding wells.

6. The method of claim 1 , wherein the pair of electrodes has the same geometric shape in every holding well.

7. The method of claim 1 , wherein at least one holding well has a pair of electrodes having a geometric shape different than the pair of electrodes positioned at an end of said plurality of holding wells.

8. The method of claim 1 , wherein said variable direct current potential source provides the same variable direct current potential to the pair of electrode ends of all holding wells.

9. The method of claim 1 , wherein said variable direct current potential source provides different variable direct current potentials to the pair of electrodes positioned at an end of different holding wells.

10. The method of claim 1 , wherein the membrane protein sample comprises solubilized membrane protein complex.

11. The method of claim 10 , wherein the membrane protein sample is within in a lipid matrix.

12. The method of claim 11 , wherein said lipid matrix has a lipid composition similar to the lipid composition of the membrane protein sample in a native environment or is varied by lipid doping.

13. The method of claim 1 , wherein said sample unit comprises a plurality of membrane protein samples of the same membrane protein.

14. The method of claim 1 , wherein said sample unit comprises a plurality of membrane protein samples of different membrane proteins.

15. The method of claim 1 , wherein said variable direct current potential source provides to the pair of electrodes positioned at an end of the holding wells a potential selected from: a subphysiological membrane potential, a physiological membrane potential, and a supra-physiological membrane potential.

16. The method of claim 1 , wherein said variable direct current potential source varies a waveform of said variable direct current potential.

17. The method of claim 11 , wherein the lipid matrix comprises a variety of lipid phases.

18. The method of claim 1 , wherein the one or more membrane proteins are comprised in a protein-detergent complex.

19. The method of claim 18 , wherein the protein-detergent complex is combined with a lipid matrix.

20. The method of claim 19 , wherein detergent diffuses from the protein-detergent complex.

Assignees

• Univ Puerto Rico

Inventors

• Lasalde-Dominicci Jose
• Quesada-Gonzalez Orestes
• Rodriguez-Cordero Josue
• Baez-Pagan Carlos
• Montoya-Zavala Martin

Classifications

• B01J2219/00585

  Parallel processes · CPC title

• B01J2219/00317

  Microwell devices, i.e. having large numbers of wells · CPC title

• B01J2219/00745

  Inorganic compounds · CPC title

• B01J2219/00725

  Peptides · CPC title

• B01L3/5085

  for multiple samples, e.g. microtitration plates · CPC title