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Structural Genomics (Protein Crystallization)

Overview of Protein Crystallization

Obtaining homogenous and monodisperse protein and subsequently crystals large enough for diffraction experiments are two of the largest barriers for crystallographers.1,2 Decreasing the amount of protein required for initial screening of crystallization conditions is desirable. Rapid crystallization associated with using ultra-small drop volumes is advantageous when screening unstable membrane proteins and protein complexes that must be purified rapidly in order to prevent degradation from endogenous proteases.3 Materials that benefit from a reduced sample requirement include the crystal screening of stable protein-protein complexes unattainable in large quantities,4 recombinant proteins expressed in cell culture, purified membrane phospholipids,5 and membrane proteins stable only in a limited set of detergents (ß-octyl glucoside, dodecyl maltoside, lauryldimethylamine oxide).6 The speed at which small crystals form can be important in the case of unstable membrane proteins that cannot remain intact for days to weeks required to perform conventional screening methods. Ink-jet technology provides the opportunity to perform initial protein crystallization screening experiments using small drops to conserve protein with high-throughput dispensing to increase the number of experiments in a given time. Small crystals achieved more rapidly during initial ink-jet screening experiments can lead the investigator to scale up using a conventional protein screening system. The successful parameters from the initial crystallization screen can then be used to obtain larger crystal targets for diffraction studies. The opportunity to utilize small crystals derived from small drops is improving as technology is currently under development for the diffraction and structural determination of microcrystals, using methods including synchrotron microfocus, atomic microscopy and nanogravimetry.7,8,9

Crystallization of Bacteriorhodopsin

A modified bicelle crystallization method for Bacteriorhodopsin (BR) developed by Faham, et al.,10, was performed using drop-on-demand ink-jet dispensing to generate crystals of BR.11,12 Bicelles are comprised of small bilayer disks, which form in lipid/amphiphile mixtures. Membrane proteins can be introduced into bicelles to experience a more bilayer like environment than what occurs in detergent micelles.13 The bicelle-forming lipid/amphiphile mixtures are more liquid at lower temperatures (< 4°C) and form a gel-like consistency at higher temperatures. Mixtures of dimyristoyl phosphotidyl choline/dihexanoyl phosphotidylcholine have a phase comprised of interconnected bicelles described as perforated lamellar structure at higher temperatures.14 It is possible to obtain various lipid bilayer structures by varying temperature of the lipid/amphiphile mixture. The low viscosity of these materials at low temperatures allows manipulation using standard screening methodology. This is not the case using the in cubo method15 in which the cubic phase is extremely viscous and would not be possible to ink-jet dispense.

BR crystals

BR crystal formation after 2 weeks in a hanging drop vapor diffusion experiment in which 100nL of BR solution was ink-jet deposited onto a 1.0 microliter drop of lipid bicelle mixture (1.0 microgram BR/drop). Crystal morphology was needle/star shaped. Maximum crystal size was 32 micrometers.

BR crystals

BR crystal formation after 2 weeks in a hanging drop vapor diffusion experiment in which 300nL of BR solution was ink-jet deposited onto a 1.0 microliter drop of lipid bicelle mixture (3.0 microgram BR/drop). Crystal morphology was rhombic star shaped. Maximum crystal size was 62 micrometers.

BR crystals

BR crystal formation after 2 weeks in a hanging drop vapor diffusion experiment in which 1000nL of BR solution was ink-jet deposited onto a 1.0 microliter drop of lipid bicelle mixture (10.0 micrograms BR/drop). Maximum crystal size was 131 micrometers.

BR crystals

Left: BR crystal formation after 2 weeks during a hanging drop vapor diffusion experiment in 1nL drop (5.0ng BR/drop) of BR/bicelle crystallization solution deposited by a cooled ink-jet device. Maximum crystal size was 20 micrometers. Right: BR crystal morphology in control sample (5.128 micrograms BR/drop). Maximum crystal size was 121 micrometers.

BR crystals

BR crystal formation in a microbatch setting under paraffin oil. 300nL of BR/bicelle was printed into a lipid droplet and subsequently mixed with a 25% concentration of the precipitant (0.6125mM NaH2PO4 pH 3.5).

Crystallization of FepA

FepA is a receptor protein in the outer membrane of E. coli that recognizes ferric enterobactin, a tricatecholate siderophore to obtain ferric ions in the growth medium. The purified FepA was a gift donation from Johann Deisenhofer of University of Texas Southwestern Medical Center.16 The protein at a concentration of 10mg/mL was mixed 1:1 with crystallization solution containing 0.1M [N-tris(hydroxymethyl)-methylglycine] (tricine) pH 8.0, 31% (w/v) PEG 1000, 0.35M NaCl, 2.5mM CaCl2, 0.055% LDAO, 15% glycerol and ink-jet deposited at 50pL, 100pL, 150pL, 50nL and 300nL onto glass coverslips. The reservoir solution contained 0.1M tricine pH 8.0, 30% PEG 1000, 0.35M NaCl, 0.06% LDAO, 10% glycerol at a volume of 300mL in the Hampton Research VDX™ hanging drop plates. The FepA crystallization solution described above was also deposited under paraffin oil at 150nL. The precipitant solution in the microbatch vapor diffusion screening experiments contained 0.1M tricine pH 8.0, 30% PEG 1000, 0.35M NaCl, 0.06% LDAO, 10% glycerol and was overprinted onto the FepA crystallization solution under Paraffin oil at 150nL. Incubation was in the dark at 20°C and 37°C. Crystals of FepA were observed after a period of three weeks in the 50pL, 100pL and 150pL drops in the hanging drop vapor diffusion experiments at 4ºC (pictures below). The concentration of protein in the drops was 0.25ng, 0.5ng, 0.75ng per 50pL, 100pL, and 150pL drop, respectively. No crystal formation has been observed in the 50nL and 300nL hanging drops or any of the drops in the microbatch experiments. The lab technician who previously crystallized the protein in Deisenhofer’s lab confirmed the FepA crystal formation.

FepA crystals

Crystals of FepA in (left to right) 150pL, 100pL, 50pL and 100pL drops after three weeks incubation at 4°C.

References

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  2. Caffrey M (2003) Membrane protein crystallization. J Struct Biol. 142:108-32.
  3. Bowie UJ (2001) Stabilizing membrane proteins. Curr Opin Struct Biol 11:397-402.
  4. Radaev S, Sun P.D. (2002) Crystallization of protein-protein complexes J. Appl. Cryst. (2002). 35, 674-676.
  5. Galdiero S, Gouaux E. (2004) High resolution crystallographic studies of alpha-hemolysin-phospholipid complexes define heptamer-lipid head group interactions: implication for understanding protein-lipid interactions. Protein Sci. 13:1503-11.
  6. McGregor CL, Chen L, Pomroy NC, Hwang P, Go S, Chakrabartty A, Prive GG. (2003) Lipopeptide detergents designed for the structural study of membrane proteins. Nat Biotechnol. 21:171-6.
  7. Pechkova I, Nicolini C (2004) Protein Nanocrystallography: new approach to structural proteomics. Trends Biotechnol. 22:117-22.
  8. Pechkova I, Nicolini C (2004) Atomic structure of a CK2alpha human kinase by microfocus diffraction of extra-small microcrystals grown with nanobiofilm template. J Cell Biochem 91:1010-20.
  9. Nicolini C, Pechkova E. (2006) Structure and growth of ultrasmall protein microcrystals by synchrotron radiation: I. microGISAXS and microdiffraction of P450scc. J Cell Biochem. 97:544-52.
  10. Faham S, Bowie JU. (2002) Bicelle crystallization: A new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure. J. Mol. Biology 316: 1-6.
  11. Howard EI, Cachau RE. (2002) Ink-jet printer heads for ultra-small-drop protein crystallography. Biotechniques. 33:1302-6.
  12. Cooley PW, Silva DS, Sprang S. (2005) Ink-jet Dispensing for Crystallization of Bacteriorhodopsin. Select Biosciences Advances in Protein Crystallography Conference, South San Francisco, California.
  13. Sanders CR, Prosser RS. (1998) Bicelles: a model membrane system for all season? Structure 6: 1227-1234.
  14. Nieh MP, Glinka JC, Krueger S, Prosser RS, Katsaras J. (2001) SANS study of the structural phases of magnetically alignable lanthanide-doped phospholipid mixtures. Langmuir 17: 2629-2638.
  15. Landau EM, Rosenbusch JP. (1996) Lipidic cubic phases: a novel concept for the crystallization of membrane proteins. Proc. Natl. Acad. Sci. USA, 93: 14532-14535.
  16. Smith BS, Kobe B, Kurumbail R, Buchanan SK, Venkatramani L, Van Der Helm D, Deisenhofer J. (1998) Crystallization and preliminary X-ray analysis of ferric enterobactin receptor FepA, an integral membrane protein from Escherichia coli. Acta Crystallogr D Biol Crystallogr. 54:697-9.