Dr.$ Darren$ E.$ Casteel$ at$ UC$ San$ Diego)$ as$ a$ template.$ The$ primers$ for$ the$ NN terminal$ends$of$all$constructs$were$designed$with$a$BamHI'site'at$their$5’$ends,$and$
the$ primers$ for$ the$ CNterminal$ ends$ were$ with$ a$NotI'site'at$ their$ 5’$ ends.$ The$
products$of$PCR$reactions$were$subjected$to$the$restriction$enzyme$treatments$after$
verifying$their$migration$patterns$on$the$1$%$agarose$gels.$$
$
Table! 2.2:! PCR! amplification! of! target! DNAs.!(A)$Composition$of$PCR$reaction$mixtures.$
(B)$Thermal$cycle$of$the$PCR$reactions.$
!
2.2.2.2$Validation$and$cleaning$of$target$DNA$
$
To$verify$the$sizes$of$the$amplified$DNA$fragments,$2$μl$of$each$PCR$reaction$
was$ loaded$ onto$ 1%$ agaroseNgel$ containing$ 1μg$ of$ ethidium$ bromide.$ The$ sizes$ of$
the$ amplified$ DNA$ fragments$ were$ verified$ by$ comparing$ their$ migration$ pattern$ to$
the$ 0.1$ ~$ 12$ kbp$ size$ marker.$ The$ DNA$ products$ were$ then$ cleaned$ using$ a$ PCR$
purification$ kit$ (Qiagen,$ Germany).$ The$ DNA$ concentrations$ were$ determined$ by$
spectrophotometric$estimation.$The$concentration$of$pure$doubleNstranded$DNA$with$
an$ A260$ of$ 1.0$ is$ 50$ μg/ml.$ Thus,$ the$ following$ formula$ was$ used$ to$ determine$ the$
DNA$concentrations:$
$
DNA$concentration$ -.
-/ = $50$3g/mL$$x$$A260$x$$dilution$factor$$$$$$$$(Equation$2.1)$$
$$
2.2.2.3$Restriction$enzyme$treatments$
!
Amplified$ target$ DNA$ and$ pQTEV$ plasmid$ were$ incubated$ with$BamHI$ and$
NotI$ as$ shown$ in$ Table$ 2.3$ (Büssow$ et$ al.,$ 2005).$ Since$ both$ restriction$ enzymes$
(B)$
Time! Temp.! No.!of!
cycles!
2$min$ 94$°C$ 1$
30$sec$ 94$°C$ $
2~3$
30$sec$ Primer$Tm$N$6~7$°C$
1~3$min$ 72$°C$
30$sec$ 94$°C$ $
28$
30$sec$ Primer$Tm$N$5~4$°C$
1~3$min$ 72$°C$
5$min$ 72$°C$ 1$
Hold$ 5$°C$ 1$
(A)$
Components! Vol./Amount!
Template$ 4$ng$
Forward$primer$(100$μM)$ 0.25$μl$
Reverse$primer$(100$μM)$ 0.25$μl$
dNTPs$mix$(25$mM)$ 1$μl$
Pfu'TaqNpolymerase$ 1$μl$
10$x$reaction$buffer$ 2.5$μl$
NucleaseNfree$water$ Add$up$to$25$μl$
show$100%$activity$in$NEBbuffer$3.1$(10$X,$NEB)$at$37°C,$the$reaction$mixtures$were$
set$up$as$a$double$digestion$method.$The$reaction$mixtures$were$incubated$at$37°C$
for$4$hr.!
Table! 2.3:! Restriction! enzyme! treatment! for! plasmid! and! insert! DNAs.$ $ (A)$ DoubleN digestion$ mixture$ for$ pQTEV$ plasmid$ DNA.$ $ (B)$ DoubleNdigestion$ mixture$ for$ a$ target$ DNA$
sequence.$$$
$
Small$byNproduct$DNA$fragments$are$a$main$cause$of$decreased$efficiency$of$
ligation$ between$ insert$ and$ vector$ DNAs.$ Therefore,$ after$ the$ restriction$ enzyme$
treatment,$ a$ gel$ DNA$ extraction$ method$ was$ applied$ to$ remove$ these$ small$ byN product$DNA$fragments.$Both$samples$were$loaded$onto$1%$agaroseNgel$containing$
1$ μg$ of$ ethidium$ bromide,$ and$ the$ gel$ containing$ the$ target$ DNA$ fragments$ were$
sliced$ out$ of$ the$ gel.$ The$ target$ DNAs$ were$ purified$ from$ the$ gel$ slices$ using$ a$
Minielute$Gel$Extraction$Kit$(Qiagen,$Germany)$according$to$its$user$manual.$
$
2.2.2.4$Ligation$
!
The$ doubleNdigested$ target$ DNA$ and$ the$ linear$ pQTEV$ were$ mixed$ with$ T4$
DNA$ ligase$ as$ shown$ in$ the$ below$ table$ below$ (Table$ 2.4)$ and$ incubated$ at$ 16°C$
overnight.$ The$ plasmid$ and$ the$ insert$ DNA$ were$ added$ in$ a$ 1:5$ molar$ ratio,$
Table!2.4:!Ligation!mixture!for!PKG!constructs.!!!
$
Components$ Vol./Amount$
Linear$pQTEV$(4.8$kbp)$ 100$ng$$
Insert$DNA$(0.5$~$2.5$kbp)$ 52$~$260$ng$$
T4$DNA$ligase$ 1$µl$
10$X$ligase$buffer$ 1$µl$
NucleaseNfree$water$ Add$up$to$10$μl$
(A) Double$digestion$reaction$for$
pQTEV$
Components Vol./Amount
pQTEV$DNA 2$μg
BamHI 1
NotI 1
10$X$NEBuffer$3.1 2 NucleaseNfree$water Add$up$to$20$μl
(B)$Double$digestion$reaction$for$the$target$
DNA$
Components Vol./Amount
pQTEV$DNA 2$μg
BamHI 1
NotI 1
10$X$NEBuffer$3.1 2 NucleaseNfree$water Add$up$to$20$μl
respectively.$ The$ amount$ of$ insert$ was$ calculated$ using$ the$ NEBioCalculatorTM$
ligation$calculator$(http://nebbiocalculator.neb.com).$
$
2.2.2.5$Transformation$
$
The$ ligated$ DNA$ was$ transformed$ into$ chemically$ competent$E.coli$ TOP10$
cells$ (Invitrogen)$ using$ the$ heat$ shock$ method.$ 2.5$ μl$ of$ the$ ligation$ mixture$ was$
mixed$ with$ 25$ μl$ of$ the$ competent$ cells$ and$ incubated$ for$ 15$ min$ on$ ice.$ The$ cells$
were$heatNshocked$at$42$°C$for$30$sec,$then$placed$on$ice$for$3$min.$PreNwarmed$LB$
medium$(1ml)$was$added,$and$the$cells$were$incubated$at$37°C$with$shacking$for$1$
hr.$Later,$the$cells$were$pelleted$by$centrifugation$at$2,300$×$g$for$10$min$and$plated$
onto$an$LB$agar$plate$containing$100$μg/ml$ampicillin$(LBAmp$agar$plate).$
$
2.2.2.6$Selection$of$positive$clone$contained$target$DNA$ $
$
The$E.coli$ containing$ pQTEV$ plasmid$ formed$ colonies$ on$ the$ LBAmp$ agar$
plate.$ Ten$ colonies$ were$ randomly$ picked$ and$ inoculated$ into$ 5$ ml$ LB$ media$
containing$100$μg/ml$ampicillin.$After$overnight$incubation$at$37°C,$the$plasmid$DNA$
was$purified$from$each$cell$culture$using$the$QIAprep$Spin$Miniprep$Kit$(Qiagen)$and$
subjected$ to$ restriction$ enzyme$ treatments$ to$ verify$ the$ insertion$ of$ the$ target$ DNA$
(Table$2.5).$The$reaction$mixtures$were$loaded$onto$1%$agarose$gel$to$verify$the$size$
of$the$insert$DNA.$
2.2.2.7$DNA$sequencing$
$
To$confirm$the$inserted$PKG$gene$sequence,$the$purified$pQTEVNPKGs$were$
sent$ to$ the$ DNA$ sequencing$ core$ at$ Baylor$ College$ of$ Medicine$ (BCM).$ The$ DNA$
Table!2.5:!Restriction!enzyme!treatment!for!insert!verification.!
Components$ Vol./Amount$
pQTEV$$ 1$μg$
BamHI' 0.5$µl$
NotI' 0.5$µl$
10$X$NEBuffer$3.1$ 1$µl$
NucleaseNfree$water$ Add$up$to10$μl$
!
sequencing$ results$ were$ examined$ using$ Basic$ Local$ Alignment$ Search$ Tool$
(BLASTJ$bl2seq)$on$the$NCBI$website$(http://blast.ncbi.nlm.nih.gov/Blast.cgi).$$
$
2.2.3$Protein$expression$and$purification$
!
2.2.3.1$Expression$and$solubility$tests$
$
The$ pQTEVNPKG$ constructs$ were$ transformed$ into$E.' coli$ TP2000$ or$ BL21$
(DE3)$ and$ tested$ resulting$ protein$ expression$ and$ solubility.$ Overexpression$ of$ the$
various$ PKG$ fragments$ was$ achieved$ by$ adding$ IPTG$ to$ a$ final$ concentration$ of$ 1$
mM.$ Three$ solution$ cultures$ were$ prepared$ to$ establish$ an$ optimal$ temperature$
condition$ of$ overexpression$ for$ each$ PKG$ construct.$ Freshly$ inoculated$E.' coli$
cultures$were$incubated$at$37°C$with$shaking$at$200$rpm$until$the$cultures$reached$
an$ optical$ density$ at$ 600$ nm$ (OD600)$ of$ 0.6$ to$ 1.0.$ IPTG$ was$ then$ added$ to$ initiate$
overexpression$of$the$PKG$proteins.$Before$induction,$1$mL$of$cell$culture$was$saved$
as$an$uninduced$control$for$SDSNPAGE$analysis.$The$cultures$containing$IPTG$were$
incubated$at$37°C$for$4$hr,$25°C$for$10$hr,$and$18$°C$for$18$hr,$respectively.$$
To$compare$the$expression$level$and$solubility$of$each$PKG$construct$using$
SDSNPAGE$(BioNrad),$0.5$mL$of$each$cell$culture$was$pelleted$and$reNsuspended$in$
100$ μL$ of$ lysis$ buffer$ (50$ mM$ potassium$ phosphate,$ 500$ mM$ NaCl,$ and$ 1$ mM$ βN mercaptoethanol$ pH$ 7.5).$ The$ reNsuspended$ cell$ samples$ were$ lysed$ by$
ultrasonication.$ For$ positive$ controls$ for$ the$ solubility$ tests,$ 40$ μL$ of$ the$ cell$ lysate$
was$ saved.$ The$ rest$ was$ centrifuged$ at$ 4°C$ at$ 15,7000$ ×$g$ for$ 30$ min$ to$ remove$
insoluble$ cell$ debris,$ and$ 40$ μL$ of$ supernatant$ was$ saved$ for$ a$ soluble$ fraction$
sample.$All$samples$were$cooked$at$95°C$for$10$min$with$1X$SDSNloading$buffer$and$
0.02$ M$ DTT.$ Then,$ 15$ μL$ of$ each$ sample$ was$ loaded$ on$ a$ 4N20$ %$ acrylamide$
gradient$SDSNPAGE$gel.$$
$
2.2.3.2$LargeJscale$protein$expression$
$
The$PKG$constructs$that$showed$robust$expression$with$high$solubility$were$
picked$ for$ largeNscale$ protein$ production$ for$ crystallization$ trials.$ 10$ mL$ of$ the$
overnight$culture$was$inoculated$in$1$liter$LB$medium$with$100$μg/ml$ampicillin$(LBAmp$
medium)$and$allowed$to$grow$at$37$°C$with$shaking$until$reaching$a$desired$OD600.$
IPTG$(final$to$0.5$mM)$was$added$to$initiate$overexpression,$and$the$cultures$were$
incubated$ in$ the$ optimal$ expression$ condition.$ After$ expression,$ the$ cells$ were$
harvested$using$centrifugation$at$4,000$×$g$for$10$min.$$
'
2.2.3.3$HisJtag$affinity$chromatography$
$
Cell$ pellets$ from$ 12$ L$ cultures$ were$ reNsuspended$ in$ 150$ mL$ of$ lysis$ buffer$
(50$mM$potassium$phosphate,$500$mM$NaCl,$and$1$mM$βNmercaptoethanol$pH$7.5)$
and$ lysed$ using$ a$ cell$ disruptor$ (Constant$ Systems,$ England).$ A$ protease$ inhibitor$
cocktail$and$100$μM$PMSF$were$added$in$the$whole$cell$lysate,$and$the$lysate$was$
centrifuged$at$maximum$125,749$×$g$for$2$hr.$The$supernatant$was$filtered$using$a$
0.22$μm$PSE$(Polyethersulfone)$bottle$top$filter.$The$supernatant$was$loaded$onto$an$
IMAC$ nickel$ column$ (BioNRad)$ on$ an$ ÄKTA$ Fast$ Flow$ Purification$ System$ (GE$
Helthcare).$The$column$was$washed$with$the$lysis$buffer$containing$30$mM$and$60$
mM$ imidazole$ before$ eluting$ the$ target$ protein.$ The$ NNterminal$ HisNtagged$ PKGs$
were$ eluted$ by$ a$ linear$ gradient$ with$ the$ lysis$ buffer$ containing$ 300$ mM$ imidazole.$
The$ eluted$ sample$ was$ dialyzed$ against$ 25$ mM$ potassium$ phosphate,$ 100$ mM$
NaCl,$1$mM$βNmercaptoethanol,$pH$7.5$overnight$at$4°C.$During$the$dialysis,$the$NN terminal$ 7×$ HisNtag$ was$ removed$ from$ the$ protein$ by$ incubating$ with$ HisNtagged$
tobacco$ etch$ virus$ protease$ (TEV)$ in$ a$ 50:1$ molar$ ratio$ (Büssow$ et$ al.,$ 2004).$ The$
sample$was$again$applied$onto$a$HiTrap$IMAC$HP$column$(5ml:$GE$healthcare)$for$
TEV$separation.$The$target$protein$without$HisNtag$was$collected$in$the$flowNthrough$
fractions.$$
$
2.2.3.4$Anion$exchange$chromatography$
$
Ion$ exchange$ chromatography$ is$ a$ method$ that$ allows$ separation$ of$
molecules$based$on$their$net$charges.$Since$all$PKG$constructs$used$for$this$study$
carry$a$net$negative$charge$at$pH$7.5$according$to$their$theoretical$isoelectric$points$
(pI)$ (Table$ 2.6),$ anion$ exchange$ chromatography$ was$ selected$ for$ further$
purification.$The$sample$was$loaded$onto$a$Mono$Q$10/100$column$(GE$Healthcare)$
to$remove$the$residual$contaminant$E.coli$proteins.$The$target$protein$was$bound$to$
the$column$with$a$binding$buffer$containing$25$mM$potassium$phosphate,$1$mM$DTT,$
pH$7.5$and$eluted$with$a$0$to$500$mM$NaCl$gradient.$The$elution$buffer$contained$1$
M$ NaCl$ in$ addition$ to$ the$ binding$ buffer.$ The$ target$ was$ confirmed$ by$ SDSNPAGE$
and$concentrated$using$a$Centricon$(size$cut$off:$10$and$30$kDa,$Millipore,$USA).!
$
2.2.3.5$Size$exclusion$chromatography$
$
The$ sample$ purification$ was$ finalized$ with$ size$ exclusion$ chromatography.$
Size$ exclusion$ chromatography$ separates$ molecules$ according$ to$ differences$ in$
native$ size.$ The$ selection$ of$ the$ size$ exclusion$ media$ depends$ on$ the$ molecular$
weight$of$the$target$protein.$In$this$study,$two$different$poreNsize$media,$Superdex$75$
and$ 200,$ were$ used$ based$ on$ the$ sizes$ of$ the$ PKG$ Iβ$ fragments$ (Table$ 2.6).$ The$
samples$ were$ loaded$ onto$ either$ HiLoad$ 16/60$ Superdex$ 75$ or$ 200$ gel$ filtration$
columns$(GE$healthcare)$equilibrated$with$a$buffer$containing$25$mM$Tris$(pH$7.5),$
150$mM$NaCl,$and$1$mM$TCEP.$$To$estimate$molecular$size$of$PKG$Iβ$proteins,$the$
molecular$size$standards$were$injected$to$the$columns.$$
$
2.2.4$X@ray$crystallography$$
!
Visualizing$ an$ atomic$ structure$ of$ macromolecules$ such$ as$ proteins,$ nucleic$
acids,$ and$ viruses$ not$ only$ provides$ detailed$ molecular$ structural$ information,$ but$
also$allow$us$to$understand$the$function$of$macromolecules$in$cells.$One$of$the$best$
tools$ to$ determine$ the$ atomic$ structure$ of$ molecules$ is$ XNray$ crystallography.$
Because$the$wavelengths$of$XNrays$are$smaller$than$those$of$visible$light$and$have$
higher$ frequency,$ XNrays$ have$ more$ energy$ to$ penetrate$ objects$ than$ visual$ light$
does.$Moreover,$the$wavelengths$of$XNrays$are$similar$to$the$radii$of$atoms$and$the$
lengths$ of$ covalent$ bonds$ (~1$ Å).$ Thus,$ XNray$ crystallography$ is$ a$ powerful$ tool$ for$
studying$macromolecules$like$protein$(Rhodes,$2006J$Rupp,$2010).$
Table! 2.6:! Basic! characteristics! of! PKG! Iβ! constructs.!Theoretical$ pI$ values$ of$ each$
construct$were$obtained$using$ProtParam$(Gasteiger$et$al.,$2005).$
Proteins$ Domains$ Amino$acid$
residue$No.$
Molecular$size$
in$monomer$
(Da)$
Theoretical$
Isoelectric$point$
(pI)$
Biochemical$
assembly$
Human$
PKG$
Full$regulatory$ 1N369$ 41645.3$ 4.80$ Dimer$
Full$regulatory$ 1N363$ 41023.6$ 4.80$ Dimer$
Full$regulatory$ 1N351$ 39688.2$ 4.87$ Dimer$
CNBNAB$ 92N369$ 31109.5$ 4.84$ Monomer$
CNBNAB$ 92N363$ 30487.8$ 4.84$ Monomer$
CNBNAB$ 92N351$ 29152.4$ 4.95$ Monomer$
CNBNA$ 92N227$ 15388.0$ 5.69$ Monomer$
CNBNB$ 217N369$ 17070.1$ 4.70$ Monomer$
$
When$ visible$ light$ (λ=$ 400$ ~$ 700$ nm)$ directly$ hits$ any$ object,$ the$ reflected$
light$bounces$off$from$the$object.$This$scattered$light$then$enters$the$lens$of$the$eye,$
and$ the$ lens$ reconstructs$ an$ image$ of$ the$ object$ and$ focuses$ it$ on$ the$ retina.$
Similarly,$when$XNrays$hit$an$object,$the$XNrays$scatter$in$certain$patterns$depending$
on$the$shape$of$molecules$consisting$the$object.$However,$the$scattered$XNrays$from$
a$single$molecule$are$too$weak$to$be$detected.$Therefore,$to$obtain$strong$diffraction$
signals$to$determine$the$structure$of$a$molecule,$many$identical$molecules$should$be$
arranged$ in$ a$ highly$ repetitive$ unit,$ called$ a$ crystal.$ Since$ lenses$ cannot$ focus$
diffracted$ XNrays$ from$ a$ crystal,$ computerNbased$ mathematical$ calculations$ are$
needed$ to$ reconstruct$ of$ an$ image$ of$ a$ protein$ molecule$ from$ XNray$ diffraction$
patterns$(Rhodes,$2006J$Rupp,$2010).$
$
2.2.4.1$Obtaining$protein$crystal:$crystallization$
!
Growing$ protein$ crystals$ is$ achieved$ by$ controlling$ the$ condition$ of$
precipitates$in$the$buffer$that$is$mixed$with$the$highly$pure$protein$sample.$There$are$
two$ main$ stages$ in$ crystallization:$ nucleation$ and$ crystal$ growth$ (Chayen$ and$
Saridakis,$ 2008J$ McPherson$ and$ Gavira,$ 2014J$ Rhodes,$ 2006).$ To$ obtain$ a$ solid$
protein$crystal,$protein$molecules$must$become$regularly$arranged$in$a$crystal$lattice,$
a$process$called$nucleation.$During$nucleation,$molecules$initially$selfNassemble$into$
clusters,$ allowing$ for$ further$ crystal$ growth.$ To$ obtain$ the$ initial$ nucleation,$ the$
protein$ sample$ must$ be$ placed$ in$ a$ chemical$ environment$ termed$ the$
! $
Figure! 2.1:! A! phase! diagram! of! macromolecules! crystallization.! Precipitant$ and$
additive$concentration,$and$buffer$pH$are$the$major$adjustable$parameters.$Modified$from$
supersaturation$zone.$This$can$be$achieved$by$decreasing$protein$solubility$using$a$
crystallization$ buffer$ condition.$ Protein$ solubility$ can$ be$ affected$ by$ several$ factors,$
such$as$concentration$of$the$protein,$nature$and$concentration$of$precipitates,$buffer$
pH,$ and$ temperature$ (Figure$ 2.1).$ Adjusting$ these$ various$ conditions$ is$ the$ key$ to$
obtaining$protein$crystals.$Several$methods$for$crystallization$are$available,$such$as$
vapor$diffusion,$microbatch,$dialysis,$and$free$interface$diffusion$(FID)$(Chayen$and$
Saridakis,$2008).$In$this$thesis,$vapor$diffusion$and$micro$batch$methods$were$used$
to$obtain$crystals.$$
!
Vapor!diffusion!!
$
Vapor$ diffusion$ is$ the$ most$ widely$ used$ method$ for$ crystallization$ of$
macromolecules.$ A$ small$ protein$ microNdroplet$ containing$ equal$ volumes$ of$ the$
protein$ and$ the$ mother$ liquor$ buffer$ is$ placed$ above$ a$ large$ volume$ of$ the$ same$
mother$ liquor$ solution$ (Figure$ 2.2).$ $ In$ this$ small$ thermodynamic$ environment,$ the$
buffer$ gradient$ between$ the$ droplet$ and$ the$ mother$ liquor$ gradually$ equilibrates$
through$ diffusion$ of$ water$ vapor$ within$ the$ system.$ Benefits$ of$ this$ method$ are$ 1)$
requiring$ very$ little$ amounts$ of$ protein$ for$ crystallization$ screening$ and$ 2)$ being$
amendable$ for$ highNthroughNput$ screening.$ Despite$ having$ these$ benefits,$ vapor$
diffusion$ method$ has$ a$ minor$ disadvantage.$ Because$ the$ crystallization$ system$
keeps$changing$during$equilibration$process,$the$protein$droplet$can$be$easily$placed$
into$unstable$conditions$where$growth$and$nucleation$are$too$rapid.$Therefore,$once$
crystals$ show$ up,$ they$ should$ be$ carefully$ monitored$ and$ frozen$ to$ avoid$ reN dissolving$or$cracking.$$
Figure!2.2:!Schematic!illustration!of!vapor!diffusion!method!with!its!phase!diagram.!!$ (A)$ Illustration$ of$ vapor$ diffusion$ method.$ A$ droplet$ of$ protein$ &$ crystallization$ buffer$
mixture$(1:1$ratio)$placed$above$the$buffer$is$initially$remained$in$an$undersaturated$zone$
(black$ dot$ in$ the$ phase$ diagram).$ (B)$ Theoretical$ phase$ diagram$ of$ vapor$ diffusion$
method.$ Vapor$ diffusion$ within$ a$ closed$ well$ system$ allows$ the$ mixture$ to$ achieve$
supersaturation$that$ facilitates$crystallization$or$ phase$ separation.$Modified$ from$(Weber,$
1997).$
Micro!batch!!
!
Crystallizing$ proteins$ with$ the$ micro$ batch$ method$ is$ achieved$ by$ mixing$
protein$samples$directly$into$crystallization$solutions$under$a$layer$of$oil$(Chayen$et$
al.,$ 1992J$ Rayment,$ 2002)$ (Figure$ 2.3).$ Several$ kinds$ of$ oil$ can$ be$ used,$ such$ as$
paraffin$ oil,$ silicon$ oil,$ or$ a$ mixture$ of$ paraffin$ and$ silicon$ oil$ (1:1$ mixtureJ$ Al’s$ oil).$
Depending$on$air$permeability$levels$of$the$oil$(paraffin$>$Al’s$>$silicon$oil),$the$water$
diffusion$ rate$ of$ the$ droplet$ can$ be$ controlled.$ In$ this$ thesis$ work,$ I$ used$ Al’s$ oil$ to$
enhance$the$vapor$diffusion$from$the$sample$to$inclease$the$crystallization.$Since$the$
oil$layer$allows$the$sample$to$more$slowly$approach$to$its$equilibrium$state$relative$to$
the$vapor$diffusion$method,$this$method$has$some$potential$to$yield$large$crystals.$$
!
2.2.4.2$Crystallization$of$CNBJA$(92J229)$of$human$PKG$Iβ$(published$in$(Kim$et$al.,$
2011))$
!
To$obtain$the$partial$apo$crystals,$the$protein$sample$was$concentrated$to$20$
mg/ml$using$a$10$kDa$cutoff$Amicon$Ultra$(Millipore).$The$initial$partial$apo$crystals$
were$ obtained$ using$ the$ vapor$ diffusion$ method$ in$ 1.4$ M$ sodium/potassium$
phosphate$(pH$5.6)$at$22$°C.$Crystal$optimization$was$done$using$an$Oryx6TM$robot$
(Douglas$ Instruments$ LTD)$ with$ the$ microbatch$ method.$ The$ biNpyramidal$ crystals$
appeared$ in$ 1.4$ M$ sodium/potassium$ phosphate$ (pH$ 8.1)$ at$ 22$ °C$ in$ 2$ days.$ CoN crystallization$with$cGMP$was$accomplished$by$adding$cGMP$(Aral$Biosynthetics)$to$
a$ final$ concentration$ of$ 5$ mM$ to$ the$ purified$ protein$ sample,$ which$ was$ then$
concentrated$to$33$mg/ml$using$a$10$kDa$cutoff$Amicon$Ultra$(Millipore).$The$crystals$
Figure!2.3:! Schematic! illustration! of! micro! batch! method!with!its!phase!diagram.!(A)$
Illustration$of$micro$batch$method.$A$protein$and$buffer$mixture$is$placed$under$a$layer$of$oil$
to$isolate$a$droplet$from$ outer$system,$preventing$rapid$evaporation.$(B)$Theoretical$phase$
diagram$of$micro$batch$method.$The$mixture$is$immediately$placed$in$supersaturation$zone$
(black$ dot$ in$ the$ phase$ diagram)$ and$ the$ mixture$ slowly$ achieves$ its$ equilibrium$ state.$
Modified$from$(Weber,$1997)$$!
of$the$PKG$Iβ:cGMP$complex$were$obtained$using$the$vapor$diffusion$method$in$0.1$
M$ sodium$ malonate$ (pH$ 5.0),$ 12%$ PEG$ 3350$ at$ 4$ °C.$ Similarly,$ coNcrystallization$
with$ cAMP$ was$ accomplished$ by$ adding$ cAMP$ to$ a$ final$ concentration$ of$ 5$ mM$ to$
the$ protein$ sample,$ which$ was$ concentrated$ with$ a$ 10$ kDa$ cutoff$ Amicon$ Ultra$
(Millipore)$to$17$mg/ml.$The$PKG$Iβ:cAMP$complex$crystals$were$obtained$using$the$
vapor$diffusion$method$in$1.4$M$sodium/potassium$phosphate$(pH$5.6)$at$4$°C.$
$
2.2.4.3$Crystallization$of$CNBJB$(217J369)$of$human$PKG$Iβ$with$cGMP$(published$in$
(Huang$et$al.,$2014))$
!
To$obtain$crystals$of$the$CNBNB:cGMP$complex,$the$protein$sample$was$preN incubated$ with$ 5$ mM$ cGMP$ and$ concentrated$ to$ 50$ mg/mL$ using$ a$ 10$ kDa$ cutoff$
Amicon$Ultra$(Millipore).$Crystals$were$obtained$using$the$hanging$drop$method.$2.5$
µL$of$protein$solution$was$mixed$with$2$µL$of$reservoir$solution$containing$1.6$M$triN sodium$ citrate$ at$ pH$ 6.5,$ and$ 0.5$ µL$ of$ 1$ M$ sodium$ iodide.$ After$ one$ week$ of$
incubation$ at$ 22$ ˚C,$ drops$ produced$ bipyramidal$ crystals$ belonging$ to$ a$ P41212$
space$ group$ that$ diffracted$ to$ 1.65$ Å$ resolution.$ Apo$ crystals$ were$ obtained$ at$ a$
much$higher$concentration$of$106$mg/mL$at$4$˚C$in$a$crystallizing$solution$containing$
25%$ (w/v)$ PEG$ 1500$ and$ 0.1$ M$ SPG$ Buffer$ pH$ 5.5.$ The$ apo$ crystal$ belongs$ to$ a$
P3121$space$group$and$diffracted$to$2.0$Å$resolution.$All$protein$concentrations$were$
measured$ using$ a$ Bradford$ assay,$ and$ all$ crystals$ were$ cryoprotected$ with$
ParatoneNN$before$freezing.$
$
2.2.4.4$Crystallization$of$CNBJAB$(92J369,$92J351)$of$human$PKG$Iβ$with$cGMP$
(Kim$et$al.$2015$manuscript)$$
!
To$obtain$crystals$of$the$human$PKG$Iβ$CNBNAB:cGMP$complex,$the$protein$
sample$was$preNincubated$with$10$mM$cGMP$and$concentrated$to$50$mg/mL$using$a$
10$ kDa$ cutoff$ Amicon$ Ultra$ (Millipore).$ Initial$ needleNshaped$ crystals$ were$ obtained$
with$the$CNBNAB$(92N351)$construct$at$22$˚C$using$the$hanging$drop$method.$0.3$µL$
of$ protein$ solution$ was$ mixed$ with$ 0.3$ µL$ of$ reservoir$ solution$ containing$ 0.2$ M$
ammonium$ sulfate,$ 20%$ PEG$ 8000,$ 10%$ Isopropanol,$ 0.1$ M$ HEPES$ at$ pH$ 7.5.$
Further$ optimization$ was$ performed$ with$ 25$ mg/ml$ protein$ concentration$ (10$ mM$
cGMP),$and$the$crystals$were$obtained$by$the$hanging$drop$method.$Drops$were$set$
up$with$2$μL$of$the$protein$solution$and$2$µL$of$reservoir$solution$containing$0.2$M$
ammonium$ sulfate,$ 17.5%$ PEG$ 8000,$ 10%$ Isopropanol,$ 0.1$ M$ HEPES$ at$ pH$ 8.0.$
After$ five$ days$ incubation$ at$ 22˚C,$ the$ drops$ produced$ big$ plateNcluster$ crystals$
belonging$to$a$C$2$2$2$space$group$that$diffracted$to$2.5$Å.$All$protein$concentrations$
were$ measured$ using$ a$ Bradford$ assay.$ Crystals$ of$ the$ cGMP$ complex$ were$
cryoprotected$with$25%$glycerol$before$freezing.$
$
2.2.4.5$Data$Collection$and$structure$determination:$Brief$theoretical$background$
!
When$ XNrays$ hit$ a$ threeNdimensional$ crystal,$ the$ XNrays$ scatter$ because$ of$
the$ electrons$ of$ the$ atoms$ in$ the$ crystal.$ These$ diffracted$ XNrays$ appear$ on$ the$
detector$as$black$spots$(reflections),$and$the$spots$include$the$information$needed$to$
reconstruct$ the$ electron$ density$ map$ of$ the$ molecule$ in$ the$ crystal,$ which$ is$ the$
ultimate$ goal$ of$ XNray$ crystallography$ (Figure$ 2.4).$ The$ diffracted$ XNrays$ undergo$
constructive$ and$ destructive$ interference$ within$ the$ crystal$ lattice,$ since$ a$ crystal$ is$
an$ ordered$ array$ of$ molecules$ (diffractions$ occur$ at$ the$ multiple$ parallel$ planes$ of$
atoms).$ Most$ of$ the$ diffracted$ XNrays$ are$ canceled$ out$ by$ destructive$ interference$
(outNofNphase),$ but$ only$ the$ diffracted$ XNrays$ fulfilling$ Bragg’s$ law$ (undergo$
constructive$interferenceJ$inNphase)$can$be$detected$as$reflections$experimentally:$$$
$
!
Figure! 2.4:! Schematic! summary! of! XQray! crystallography.!Once$ a$ three$ dimensional$
crystal$is$exposed$to$attenuated$XNray$beam,$the$scattered$XNray$beam$results$in$diffraction$
patterns$ (reflections)$ with$ different$ intensities$ on$ detector,$ which$ contains$ information$ of$
molecules$ in$ the$ crystal.$ These$ reflections$ represent$ positions$ of$ atoms$ in$ a$ reciprocal$
space,$ which$ is$ Fourier$ transformation$ (FT)$ of$ the$ crystal$ lattice$ points$ in$ a$ real$ space.$ In$
order$ to$ reconstruct$ electron$ density$ map$ (?(A);$real$ space$ map),$ phase$ information$ must$
be$deduced$by$experimentally$or$molecular$replacement$with$inverse$Fourier$transformation$
(FTN1).$ Then,$ a$ model$ can$ be$ built$ based$ on$ the$ electron$ density$ map.!Modified$ from$
(Hesegawa,$2012)
DE$ = $2FsinH$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$(Equation$2.2)$$$$$
$
where$D$is$an$integer,$E$is$the$wavelength$of$incident$XNray,$H$is$the$incident$angle$of$
the$XNray,$and$F$is$the$distance$between$the$repeating$planes$in$the$crystal$(Figure$
2.5).$If$the$wavelength$(E)$and$the$diffraction$angle$(H)$are$given,$the$distance$(F)$can$
be$calculated,$based$on$Bragg’s$law.$This$means$that$Bragg’s$law$allows$us$to$obtain$
a$ measure$ of$ the$ distance$ between$ the$ lattice$ planes$ in$ the$ crystal$ from$ the$ XNray$
scattering,$ so$ the$ crystal$ lattice$ (unit$ cell$ parameters)$ can$ be$ deduced$ from$ the$
diffraction$ pattern$ (Rhodes,$ 2006J$ Rupp,$ 2010).$ Because$ the$ diffraction$ image$ is$ a$
snapshot$of$the$planes$in$the$crystal$that$satisfy$Bragg’s$law,$the$crystal$needs$to$be$
rotated$during$collecting$diffraction$images$to$obtain$the$information$of$the$full$set$of$
the$lattice$planes$in$the$crystal.$
$
While$the$position$of$the$reflection$is$independent$of$the$position$of$the$atoms$
in$a$unit$cell$(only$relied$on$the$space$group$and$the$unit$cell$dimension),$the$intensity$
of$the$reflections$contains$the$information$of$all$atomic$arrangement$in$the$unit$cell.$
Although$ the$ scattering$ intensity$ is$ readily$ obtained$ from$ an$ XNray$ diffraction$
experiment,$ the$ relative$ phase$ information$ cannot$ be$ determined$ during$ data$
collection.$Since$the$electron$density$map$cannot$be$obtained$without$the$phase,$the$
phase$determination$is$an$essential$step$to$solving$the$crystal$structure.$This$is$called$
the$ “phase$ problem”$ for$ XNray$ crystallography$ (Figure$ 2.4).$ $ Since$ the$ intensity$
! !
Figure! 2.5:! Illustration! of! Bragg’s! raw.!When$incident$XNray$beams$with$an$angle$of$L$ hit$ an$ array$ of$ atoms$ on$ multiple$ parallel$ planes$ of$ a$ crystal,$ the$ beams$ only$ satisfies$
Bragg’s$raw$can$be$scattered$with$the$same$angle$L.$$Based$on$Bragg’s$raw,$a$distance$
between$ parallel$ planes$and$ arrays$of$the$atoms$(unit$cell$parameters)$can$be$deduced.$
Modified$from$(Serdyuk$et$al.,$2007).!
!