pre - crystallization lcp - frap assays are optional ; however , they can significantly accelerate the process of searching for initial crystallization conditions , especially in the case of difficult membrane proteins . purify a membrane protein of interest in a detergent solution and concentrate the protein / detergent complexes to ~10 - 20 mg / ml , taking care not to over - concentrate the detergent .
transfer ~25 mg of an lcp host lipid ( typically monoolein ) or a lipid mixture into a 1.5 ml plastic tube and incubate at 40 c for few minutes until the lipid melts .
load another 100 l syringe with the protein solution at a protein solution - to - lipid ratio 2/3 v / v .
push the syringe plungers alternately to move the lipid and protein through the inner needle of the coupler , back and forth , until the lipid mesophase becomes homogeneous .
lcp forms spontaneously upon mechanical mixing , and the protein becomes reconstituted in the lipid bilayer of lcp .
formation of lcp can be verified by its transparent and gel - like consistency and by the absence of birefringency when viewed under a microscope equipped with cross - polarizers , or , if possible , by using small - angle x - ray diffraction .
lcp - frap assays are designed to measure the diffusion properties of membrane proteins reconstituted in lcp at a variety of screening conditions .
the long - range diffusion of membrane proteins in lcp is essential for successful crystallization ; however , the microstructure of lcp constrains diffusion of large proteins or oligomeric protein aggregates .
a common reason for failure of an in meso crystallization experiment is a fast protein aggregation leading to a loss of diffusion .
it has been shown that the aggregation behavior of a protein depends on the particular protein construct , the host lipid and the composition of the screening solution .
label the protein with a fluorescent dye ( cy3 or similar ) at a protein / dye ratio of ~100/1 , remove the unreacted dye and concentrate the protein to ~ 1 mg / ml .
when labeling free amines , use ph between 7 and 7.5 to predominately label the free n - terminus .
be aware that amino labeling can also label lipids co - purified with the protein .
reconstitute the labeled protein in lcp as described in section 1 ) . set up assay plates as described in section 3 ) using lcp - frap screening solutions instead of crystallization screens .
incubate the plates at 20 c in the dark for at least 12 hours to achieve an equilibrium state .
place one of the plates on the lcp - frap station and focus on the first well using a 10x objective .
trigger the laser . the laser power and number of pulses should be adjusted to bleach ~30 - 70% of the labeled protein in the middle of the bleached spot . immediately after triggering the laser , start recording a fast post - bleaching sequence of ~200 images at the fastest possible rate . follow with recording of a slow post - bleach sequence of ~50 images , selecting the delay between images as 1 - 20 s , depending on the diffusion rate of the protein . integrate the intensity inside the bleach spot in all frames and correct it for bleaching and light intensity fluctuations during the acquisition by dividing the intensity inside the bleached spot by the averaged intensity of a reference spot outside of the laser bleached area . normalize the corrected intensity to make the pre - bleached intensity equal to 1 and the initial bleached intensity equal to 0 .
fit the curve of the normalized intensity vs. time , f(t ) , using the following equation : f(t ) = m x exp(-2t / t ) x ( i0(2t / t ) + i1(2t / t ) ) , ( eq.1 ) where m is the mobile fraction of diffusing molecules , t is the characteristic diffusion time , t is the real time of each recorded frame , i0 and i1 are the 0 and 1 order modified bessel functions . calculate the diffusion coefficient , d , as : d = r/4 t , ( eq.2 ) where r is the radius of the bleached spot .
design new crystallization screens based on the components that facilitated protein diffusion and excluding conditions for which protein diffusion was not observed . if the protein did not diffuse in any of the screened conditions , consider broadening the screening space or trying a new protein construct .
transfer the protein - laden lcp into a 10 l gas - tight syringe attached to a repetitive syringe dispenser .
attach a short removable needle ( gauge 26 , 10 mm length ) to the 10 l syringe .
dispense 200 nl boluses of lcp on the surface of four adjacent wells forming a 2x2 square .
repeat steps 3.4)-3.6 ) with the next set of 4 wells until the whole plate is filled .
incubate the plate at a constant temperature , periodically checking for crystal formation and growth .
place a plate with protein crystals under a stereo microscope with variable zoom , equipped with a linear rotating polarizer and analyzer .
focus on the well of interest using a low power zoom so that the whole well is placed within the field of view .
score the coverslip glass in four strokes making a square inside the well boundaries using a sharp corner of a ceramic capillary cutting stone .
press around the scored perimeter with strong sharp - point tweezers to propagate the scratches through the thickness of the coverslip glass .
inject few l of precipitant solution through one of the holes to reduce dehydration during the subsequent steps . using an angled sharp needle probe break up the glass along one or two sides to free the cut - out square .
if the bolus is stuck to the coverslip , then flip the glass square over and place on the bottom of the well . add an extra few l of precipitant solution , supplemented with a cryo - protectant , if necessary , on top of the exposed cubic phase bolus in the well .
adjust the angle between the polarizer and the analyzer to increase the contrast between the birefringent crystal and the background , while keeping enough light to see the harvesting loop .
select a mitegen micromount with a diameter matching the crystal size and then harvest the crystal directly from the lcp by scooping it into the micromount .
flash freeze the micromount with the harvested crystal in liquid nitrogen , and ship it to a synchrotron source beamline for x - ray data collection .
an engineered human beta 2 adrenergic g protein - coupled receptor ( 2ar - t4l ) was expressed in baculovirus infected sf9 insect cells and purified in dodecylmaltoside ( ddm)/ cholesteryl hemisuccinate ( chs ) detergent solution bound to a partial inverse agonist carazolol .
the protein was labeled with cy3 nhs ester and used in lcp - frap pre - crystallization assays ( figure 2 ) .
coarse grid screens based on several conditions selected from the results of lcp - frap assays produced initial crystal - like hits ( figure 3 ) .
a ) results of an lcp - frap assay performed in an automatic high - throughput mode , in which each sample of a 96-well plate is bleached sequentially and fluorescence recovery is measured after a 30 min incubation .
the obtained fluorescence recoveries , which represent the mobile fraction in each sample , are plotted for all 96 samples .
the screening solutions contain 0.1 m tris ph 8 , 30 % v / v peg 400 combined with 48 different salts at two different concentrations .
1.the mobile fractions and the diffusion coefficients are determined using eqs . 1 and 2 .
fast recovery of less than 10% in the sample containing na chloride is due to fluorescently labeled lipids co - purified with the protein .
initial crystal hits of 2ar - t4l / carazolol obtained by a coarse grid screening around most promising conditions identified by lcp - frap , containing na sulfate ( panel a ) and na formate ( panel b ) .
the protein is labeled with cy3 nhs ester and the fluorescent images are taken using excitation at 543 nm and emission at 605 nm .
the images of crystals grown in the presence of na sulfate ( panels a and b ) and k formate ( panels c and d ) are taken in the brightfield mode ( panels a and c ) and using cross - polarizers ( panels b and d ) .
purify a membrane protein of interest in a detergent solution and concentrate the protein / detergent complexes to ~10 - 20 mg / ml , taking care not to over - concentrate the detergent .
transfer ~25 mg of an lcp host lipid ( typically monoolein ) or a lipid mixture into a 1.5 ml plastic tube and incubate at 40 c for few minutes until the lipid melts .
load another 100 l syringe with the protein solution at a protein solution - to - lipid ratio 2/3 v / v .
push the syringe plungers alternately to move the lipid and protein through the inner needle of the coupler , back and forth , until the lipid mesophase becomes homogeneous .
lcp forms spontaneously upon mechanical mixing , and the protein becomes reconstituted in the lipid bilayer of lcp .
formation of lcp can be verified by its transparent and gel - like consistency and by the absence of birefringency when viewed under a microscope equipped with cross - polarizers , or , if possible , by using small - angle x - ray diffraction .
lcp - frap assays are designed to measure the diffusion properties of membrane proteins reconstituted in lcp at a variety of screening conditions .
the long - range diffusion of membrane proteins in lcp is essential for successful crystallization ; however , the microstructure of lcp constrains diffusion of large proteins or oligomeric protein aggregates .
a common reason for failure of an in meso crystallization experiment is a fast protein aggregation leading to a loss of diffusion .
it has been shown that the aggregation behavior of a protein depends on the particular protein construct , the host lipid and the composition of the screening solution .
label the protein with a fluorescent dye ( cy3 or similar ) at a protein / dye ratio of ~100/1 , remove the unreacted dye and concentrate the protein to ~ 1 mg / ml .
when labeling free amines , use ph between 7 and 7.5 to predominately label the free n - terminus .
be aware that amino labeling can also label lipids co - purified with the protein .
reconstitute the labeled protein in lcp as described in section 1 ) . set up assay plates as described in section 3 ) using lcp - frap screening solutions instead of crystallization screens .
incubate the plates at 20 c in the dark for at least 12 hours to achieve an equilibrium state .
place one of the plates on the lcp - frap station and focus on the first well using a 10x objective .
trigger the laser . the laser power and number of pulses should be adjusted to bleach ~30 - 70% of the labeled protein in the middle of the bleached spot . immediately after triggering the laser , start recording a fast post - bleaching sequence of ~200 images at the fastest possible rate . follow with recording of a slow post - bleach sequence of ~50 images , selecting the delay between images as 1 - 20 s , depending on the diffusion rate of the protein . integrate the intensity inside the bleach spot in all frames and correct it for bleaching and light intensity fluctuations during the acquisition by dividing the intensity inside the bleached spot by the averaged intensity of a reference spot outside of the laser bleached area . normalize the corrected intensity to make the pre - bleached intensity equal to 1 and the initial bleached intensity equal to 0 .
fit the curve of the normalized intensity vs. time , f(t ) , using the following equation : f(t ) = m x exp(-2t / t ) x ( i0(2t / t ) + i1(2t / t ) ) , ( eq.1 ) where m is the mobile fraction of diffusing molecules , t is the characteristic diffusion time , t is the real time of each recorded frame , i0 and i1 are the 0 and 1 order modified bessel functions . calculate the diffusion coefficient , d , as : d = r/4 t , ( eq.2 ) where r is the radius of the bleached spot .
design new crystallization screens based on the components that facilitated protein diffusion and excluding conditions for which protein diffusion was not observed .
if the protein did not diffuse in any of the screened conditions , consider broadening the screening space or trying a new protein construct .
reconstitute the protein in lcp as described in section 1 ) . transfer the protein - laden lcp into a 10 l gas - tight syringe attached to a repetitive syringe dispenser . attach a short removable needle ( gauge 26 , 10 mm length ) to the 10 l syringe .
dispense 200 nl boluses of lcp on the surface of four adjacent wells forming a 2x2 square .
apply a gentle pressure on the coverslip to seal the wells . repeat steps 3.4)-3.6 ) with the next set of 4 wells until the whole plate is filled .
incubate the plate at a constant temperature , periodically checking for crystal formation and growth .
place a plate with protein crystals under a stereo microscope with variable zoom , equipped with a linear rotating polarizer and analyzer .
focus on the well of interest using a low power zoom so that the whole well is placed within the field of view .
score the coverslip glass in four strokes making a square inside the well boundaries using a sharp corner of a ceramic capillary cutting stone .
press around the scored perimeter with strong sharp - point tweezers to propagate the scratches through the thickness of the coverslip glass .
punch two small holes at opposite corners of the scored square . inject few l of precipitant solution through one of the holes to reduce dehydration during the subsequent steps . using an angled sharp needle probe break up the glass along one or two sides to free the cut - out square .
if the bolus is stuck to the coverslip , then flip the glass square over and place on the bottom of the well . add an extra few l of precipitant solution , supplemented with a cryo - protectant , if necessary , on top of the exposed cubic phase bolus in the well .
adjust the angle between the polarizer and the analyzer to increase the contrast between the birefringent crystal and the background , while keeping enough light to see the harvesting loop . select a mitegen micromount with a diameter matching the crystal size and then harvest the crystal directly from the lcp by scooping it into the micromount .
flash freeze the micromount with the harvested crystal in liquid nitrogen , and ship it to a synchrotron source beamline for x - ray data collection .
an engineered human beta 2 adrenergic g protein - coupled receptor ( 2ar - t4l ) was expressed in baculovirus infected sf9 insect cells and purified in dodecylmaltoside ( ddm)/ cholesteryl hemisuccinate ( chs ) detergent solution bound to a partial inverse agonist carazolol .
the protein was labeled with cy3 nhs ester and used in lcp - frap pre - crystallization assays ( figure 2 ) .
coarse grid screens based on several conditions selected from the results of lcp - frap assays produced initial crystal - like hits ( figure 3 ) .
a ) results of an lcp - frap assay performed in an automatic high - throughput mode , in which each sample of a 96-well plate is bleached sequentially and fluorescence recovery is measured after a 30 min incubation .
the obtained fluorescence recoveries , which represent the mobile fraction in each sample , are plotted for all 96 samples .
the screening solutions contain 0.1 m tris ph 8 , 30 % v / v peg 400 combined with 48 different salts at two different concentrations .
1.the mobile fractions and the diffusion coefficients are determined using eqs . 1 and 2 .
fast recovery of less than 10% in the sample containing na chloride is due to fluorescently labeled lipids co - purified with the protein .
initial crystal hits of 2ar - t4l / carazolol obtained by a coarse grid screening around most promising conditions identified by lcp - frap , containing na sulfate ( panel a ) and na formate ( panel b ) .
the protein is labeled with cy3 nhs ester and the fluorescent images are taken using excitation at 543 nm and emission at 605 nm .
the images of crystals grown in the presence of na sulfate ( panels a and b ) and k formate ( panels c and d ) are taken in the brightfield mode ( panels a and c ) and using cross - polarizers ( panels b and d ) .
the protocols provide a basic visual guidance for the main steps involved in performing in meso crystallization experiments .
more in - depth details related to these protocols , emphasizing possible pitfalls , shortcomings or alternative routes are available elsewhere .
optional lcp - frap assays can help at the earlier stages to select the most promising protein construct , lcp host lipid and lipid additives , as well as limit the range of possible precipitants and buffer conditions .
once an initial crystallization hit is found , it should be optimized to obtain better quality crystals .
optimization of in meso crystallization conditions is essentially similar to optimizing conditions for soluble proteins with the addition of extra parameters associated with the composition of lcp .
membrane protein crystals grown in lipidic mesophase are typically smaller in size than crystals obtained in detergent solution , but more ordered , thus , benefitting strongly from using microfocus beamlines available at modern synchrotron sources .
many of the procedures related to in meso crystallization , including setting up crystallization or assay plates , conducting lcp - frap assays and detecting crystals , have been semi- or fully automated , allowing screening of a large range of conditions while consuming small amounts of protein and lipid . on the other hand , protein reconstitutions in lcp and crystal harvesting remain manual and more tedious operations and , thus , have a need for improvement .
 membrane proteins perform critical functions in living cells related to signal transduction , transport and energy transformations , and , as such , are implicated in a multitude of malfunctions and diseases . however , a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners , mainly , due to difficulties associated with their solubilization and generation of diffraction quality crystals . 
 crystallization in lipidic mesophases ( also known as in meso or lcp crystallization ) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins , photosynthetic proteins , outer membrane beta barrels and g protein - coupled receptors . in meso 
 crystallization takes advantage of a native - like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions . 
 the method is not difficult , but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials . 
 here we demonstrate a simple and efficient way of making lcp and reconstituting a membrane protein in the lipid bilayer of lcp using a syringe mixer , followed by dispensing nanoliter portions of lcp into an assay or crystallization plate , conducting pre - crystallization assays and harvesting crystals from the lcp matrix . 
 these protocols provide a basic guide for approaching in meso crystallization trials ; however , as with any crystallization experiment , extensive screening and optimization are required , and a successful outcome is not necessarily guaranteed . 
Protocol
1. Protein Reconstitution in LCP
2. LCP-FRAP Pre-crystallization Assays
3. Setting Up LCP Crystallization Trials
4. Harvesting Crystals from LCP
5. Representative Results:
Discussion
Disclosures