Use of dye to distinguish salt and protein crystals under microcrystallization conditions

• US 20070111317A1
• Filed: 12/15/2006
• Published: 05/17/2007
• Est. Priority Date: 07/30/2001
• Status: Abandoned Application

• Current Assignee: UAB Research Foundation (University of Alabama System)
• Original Assignee: UAB Research Foundation (University of Alabama System)
• Inventors: Gester, Thomas, Hamrick, David, Bray, Terry, Cosenza, Larry, Delucas, Lawrence
• Application Number:
  US11/639,210
• Publication Number:
  US 20070111317A1
• Time in Patent Office: Days
• US Class Current: 436/86
• CPC Class Codes: C07K 1/306 by crystallization
• CPC Class Codes: C30B 29/58 Macromolecular compounds
• CPC Class Codes: C30B 7/00 Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions from molten solvents C30B9/00; by normal or gradient freezing C30B11/00; under a protective fluid C30B27/00
• CPC Class Codes: G01N 25/147 by cristallisation
• CPC Class Codes: G01N 33/00 Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
• CPC Class Codes: G01N 33/6839 involving dyes, e.g. Coomassie blue, bromcresol green

Abstract

An improved method of screening crystal growth conditions is provided wherein molecules are crystallized from solutions containing dyes. These dyes are selectively incorporated or associated with crystals of particular character thereby rendering crystals of particular character colored and improving detection of the dyed crystals. A preferred method involves use of dyes in protein solutions overlayed by oil. Use of oil allows the use of small volumes of solution and facilitates the screening of large numbers of crystallization conditions in arrays using automated devices that dispense appropriate solutions to generate crystallization trials, overlay crystallization trials with an oil, provide appropriate conditions conducive to crystallization and enhance detection of dyed (colored) or undyed (uncolored) crystals that result.

Claims

1-54. -54. (canceled)

55. A method for screening dyed protein crystals comprising:

(a) providing a protein solution, wherein the protein solution contains at least one dye, which dye is capable of dyeing at least a portion of protein crystals formed from the protein solution;

(b) subjecting the protein solution to environmental conditions effective to form protein crystals;

(c) detecting the presence of dyed protein crystals, whereby the presence of the dyed protein crystals indicates the presence of protein crystals; and

(d) subjecting the protein crystals to x-ray diffraction analysis.

56. The method of claim 55, wherein the dye is selected from the group consisting of methylene blue, methylene green, Izit and crystal violet.

57. The method of claim 55, wherein the dye binds to protein molecules of the protein crystals formed from the protein solution.

58. The method of claim 55, wherein the dye does not bind to protein molecules of the protein crystals formed from the protein solution and wherein molecules of the dye reside in channels present in the protein crystals.

59. The method of claim 55, wherein the protein solution is partitioned from the atmosphere.

60. The method of claim 55, wherein the protein solution further comprises a component selected from the group consisting of salts, buffers, precipitants, crystallization aids and any combination thereof.

61. A method for screening protein crystal growth conditions, comprising:

(a) providing a set of at least two protein solutions, wherein the protein solutions contain a dye, which dye (i) is capable of dyeing at least a portion of protein crystals formed from the protein solution and (ii) does not dye a significant portion of component crystals formed from the protein solution;

(b) subjecting the protein solutions to predetermined conditions, wherein the environmental conditions to which each member of the set of protein solutions is subjected is not identical to the environmental conditions to which another member of the set is subjected;

(c) detecting the presence or non-presence of dyed protein crystals and/or undyed component crystals, wherein;

(i) the presence of the dyed protein crystals indicates the formation of protein crystals; and

(ii) the presence of undyed component crystals indicates formation of component crystals; and

(d) subjecting the protein crystals to x-ray diffraction analysis.

64. The method of claim 61, wherein the dye is selected from the group consisting of methylene blue, methylene green, Izit and crystal violet.

65. The method of claim 61, wherein the protein solutions are partitioned from the atmosphere.

66. The method of claim 61, wherein the protein solution further comprises a component selected from the group consisting of salt, buffer, precipitant, crystallization aid and any combination thereof.

62. The method of 61, wherein the set of protein solutions consists of greater than 10 protein solutions.

63. The method of 62, wherein the set of protein solutions consists of greater than 100 protein solutions.

67. A method for screening dyed component crystals comprising

(a) providing a component solution, wherein the component solution contains at least one dye, which dye is capable of dyeing at least a portion of component crystals formed from the component solution;

(b) subjecting the component solution to environmental conditions effective to form component crystals;

(c) detecting the presence of dyed component crystals, whereby the presence of the dyed component crystals indicates the presence of component crystals; and

(d) subjecting the component crystals to x-ray diffraction analysis.

68. The method of claim 67, wherein the component solution is partitioned from the atmosphere.

69. The method of claim 67, wherein the component solution further comprises a protein.

70. The method of claim 69, wherein the protein crystallizes under the environmental conditions of (b).

71. The method of claim 70, wherein step (b) comprises addition of a precipitate solution.

72. The method of claim 70, wherein the protein crystals comprises undyed protein crystals.

73. An automated method of screening protein crystal growth conditions comprising the steps of:

(a) providing a set of at least two protein solutions, wherein the protein solutions contain a dye, which dye (i) is capable of dyeing at least a portion of protein crystals formed from the protein solution and (ii) does not dye a significant portion of component crystals formed from the protein solution;

(b) dispensing the protein solutions onto a platform, wherein the protein solutions are dispensed using an automated dispensing system in accordance with a predetermined program;

(c) controlling the protein crystal growth conditions of the protein solutions;

(d) detecting the presence or non-presence of dyed protein crystals and/or undyed component crystals, wherein the presence of the dyed protein crystals indicates the formation of protein crystals; and

the presence of undyed component crystals indicates the formation of component crystals; and

(e) subjecting the protein crystals to x-ray diffraction analysis.