g protein - coupled receptors 
 ( gpcrs ) are integral membrane proteins 
 that transduce extracellular signals such as light , hormones , and 
 chemoattractants to downstream signal pathways .
activated gpcrs are phosphorylated by a family of serine / threonine 
 kinases named g protein - coupled receptor kinases ( grks ) , a process 
 that initiates their desensitization .
the ability to interact with 
 membranes in which gpcrs are found is essential for grk function .
grk1 and grk7 are 
 post - translationally modified by prenyl groups at their c - termini . 

grk2 and grk3 , on the other hand , bind to membranes by virtue of their 
 c - terminal pleckstrin homology ( ph ) domains , which bind to acidic 
 phospholipids and interact with heterotrimeric g subunits , 
 which are prenylated .
grk4 , grk5 , and grk6 constitute a subfamily 
 of grks that have 23 membrane - binding motifs .
the first is 
 a basic segment near the n - terminus that is believed to be responsible 
 for binding phosphatidylinositol-4,5-bisphosphate ( pip2 ) .
the second is an amphipathic helix located at the extreme c - terminus 
 that is believed to interact with anionic lipid bilayers .
grk4 and 
 grk6 are in addition palmitoylated on cysteines immediately c - terminal 
 to this helix .
the n - terminal 18 amino acids of all grks , 
 however , are highly conserved , predicted to have helical propensity , 
 and are essential for phosphorylation of activated gpcrs .
proposed 
 roles for these residues include either direct interaction with activated 
 receptors or with the phospholipid bilayer , either of which is proposed to induce helical character in this 
 region and promote the formation of an activated form of the kinase 
 domain . over the past several years
, crystallographic studies 
 have yielded 
 new insights into the molecular mechanism for regulation of grks by 
 their interactions with receptors and membranes .
however , crystallographic analysis requires the removal 
 of protein complexes from their native membrane environment and can not 
 provide direct information on how these molecules are arranged on 
 the membrane surface in situ .
sum frequency generation 
 ( sfg ) vibrational spectroscopy is a powerful tool to examine peptides 
 and proteins at biointerfaces such as lipid bilayers .
for example , orientations of peptides with different secondary structures , 
 such as linear -helices , bent -helices , -sheets , and 310-helices associated with solid substrate supported lipid 
 bilayers have been deduced using polarized sfg studies .
sfg has also 
 been applied to investigate the membrane orientations of g , 
 the ggrk2 complex , and g 
 heterotrimers in situ .
recently , 
 we showed that the use of both sfg and attenuated total reflectance 
fourier 
 transform infrared ( atr - ftir ) spectroscopy can determine orientations 
 of complex proteins with greater certainty . in this research , we used sfg and atr - ftir to study the membrane 
 interactions of the n- and c - terminal segments of grk5 to gain insight 
 into which regions were most important for membrane binding and what 
 structure and orientation they adopt while interacting with membranes
. grk5 residues 231 ( grk5231 ) are highly 
 conserved in the grk4 subfamily of grks ( figure 1 ) , which includes grk4 , grk5 , and grk6 . in previous research , it 
 was suggested that residues 2229 , which include basic amino 
 acids lys22 , arg23 , lys24 , lys26 , lys28 , and lys29 , bind to pip2 . an overlapping region ( residues 
 2039 ) has also been implicated in binding to calmodulinca ( camca ) .
the structure of grk6 ( a close homologue of grk5 ) determined by 
 x - ray crystallography suggests that the n - terminal portion of the 
 peptide ( residues 223 ) is disordered when the enzyme is in 
 an inactive state , but residues 218 
 become ordered when the enzyme assumes a more active , presumably receptor - bound 
 conformation .
however , it is not known 
 if this region forms a platform for binding to lipid membranes or 
 activated gpcrs .
therefore , elucidating the ability of different segments 
 of the grk5 n - terminus to interact with the membrane is key to understanding 
 how the membrane influences grk5 function .
sequences of the human 
 grk5 n - terminal and c - terminal peptides 
 used in this study .
residues highlighted in gray adopt an -helical 
 conformation in the structure of the grk6sangivamycin complex .
the c - terminal residues 552562 of grk5 are believed 
 to 
 be another region that interacts with phospholipids .
residues 549557 are predicted to form 
 an amphipathic helix when bound to membranes . in the active conformation of the grk6 crystal structure ,
residues 
 548557 form an amphipathic helix that docks to the core of 
 the enzyme but is far removed from the predicted membrane surface 
 and the n - terminal segment believed to bind pip2 .
thus , either this structural element does not 
 bind to membranes , or it only binds to membranes when grk6 is in a 
 more inactive state , or the structure represents a soluble form of 
 the enzyme , such as when it translocates to the nucleus to phosphorylate 
 transcription factors . by combining 
 data from two complementary optical spectroscopic 
 techniques , sfg and atr - ftir , we are seeking to answer the following 
 questions .
first , do peptides representing the n - terminal ( grk5231 ) and c - terminal ( grk5546565 ) 
 regions bind to membranes on their own , and , if so , what structure 
 do they adopt ?
finally , is camca able to 
 dissociate these grk5 peptides from the membrane , as proposed to be 
 required for nuclear translocation ?
peptides grk5231 , grk5224 , and grk5546565 ( figure 1 ) were synthesized by the following procedure .
protected 
 amino acids and n - methylpyrrolidone ( nmp ) , 1-hydroxybenzotriazole 
 ( hobt ) , and o - benzotriazole - n , n , n,n-tetramethyluronium 
 hexafluorophosphate ( hbtu ) were purchased from creosalus .
acetonitrile , 
 hplc grade water , trifluoroacetic acid ( tfa ) , diethyl ether , and phenol 
 were from fisher scientific .
piperidine , n , n - diisopropylethylamine 
 ( dipea ) , dimethylformamide ( dmf ) , thioanisole , triisopropylsilane 
 ( tips ) , and calmodulin were from sigma - aldrich . solid - phase synthesis 
 resin novapeg rink amide resin ( 0.5 mmol / g ) was purchased from novabiochem . 

analytical hplc analysis was done using an alliance system with 250 
  5 mm c18 3 m column ( vydac ) .
mass spectrometry analysis 
 was done using a 6130 quadrupole lc / ms ( agilent technologies ) .
semipreparative 
 hplc purification was performed using a delta 600 system ( waters ) 
 with 150  19 mm xbridge prep c18 10 m obd column ( waters ) . 

hplc analysis and purification were done using solvent system 0.1% 
 tfa in water and 0.1% tfa in acetonitrile .
the syntheses of c - terminal sequences up to ala were carried out on a cs336x automated synthesizer 
 ( c.s .
bio co. ) , and the syntheses were then continued on a discover 
 sps single mode manual microwave synthesizer ( cem corp . ) ( power = 
 20 w , 5 min per coupling and power 20 w , 1.5 min per deprotection ; 
 temperature 7075 c ) .
the general protocol included double coupling and double deprotection 
 as well as acetylation of the unreacted amino groups .
coupling cycles 
 were performed using 4 equiv of incoming amino acid , hobt / hbtu in 
 dmf and dipea in nmp .
cleavage of the peptide from the resin 
 and side - chain deprotection was performed using 10 ml of the mixture 
 di water : phenol : thioanisole : tips : tfa ( 0.5 ml:0.7 g:0.5 ml:0.25 ml:8.75 
 ml ) .
the reaction was left running at room temperature for 2 h. after 
 filtration of the resin , crude peptide was precipitated with cold 
 ethyl ether .
the purity of the final peptide was analyzed 
 using hplc and molecular weight confirmed by ms .
popc ( 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine ) 
 and pip2 were purchased from avanti polar lipids . supported popc / popc lipid bilayers 
 were constructed on caf2 prisms by the langmuir 
the first popc layer is deposited on one of the square faces of 
 the right - angle caf2 prism with a ksv2000 lb system : the 
 plasma - cleaned prism was first immersed in the lb trough .
then a certain 
 amount of popc chloroform solution , typically 5 drops of 10 mg / ml , 
 was spread on the water surface until the surface tension reaches 
 5 mn / m .
two barrier arms were suppressed so that the surface 
 tension remained 34 mn / m , while the caf2 prism was lifted 
 from the subphase .
a layer of popc lipids was deposited on the face 
 perpendicular to the water surface in this way . after aligning the 
 laser beams to find the monolayer signal , a 2 ml reservoir filled 
 with water was placed beneath the prism .
popc lipids were added to 
 the surface of the water in the reservoir so that the surface tension 
 was around 34 mn / m .
the reservoir was elevated so that the the lipid 
 monolayer on the water surface contacted with the first layer deposited 
 on the prism to form a lipid bilayer .
sfg theory , our experimental design , and data analysis method have been reported before .
the concentration of each of the four 
 peptides was 3.8 m , and the peptides were dissolved in 10 mm 
 potassium phosphate buffer ( ph 7.4 ) . because caf2 prisms 
 were used as substrates to prepare the lipid bilayers , small amounts 
 of ca may be dissolved in the subphase .
2 mm edta was 
 added to the above buffer solution to minimize any influence of the 
 ca released from the caf2 substrates . for 
 each of the three n - terminal peptides studied here
, we added the peptide 
 into the subphase in contact with the substrate supported bilayer 
 and after equilibration recorded the sfg signal in the water o  h 
 stretching frequency range as well as in the peptide amide i frequency 
 region .
for all peptides we studied , the adsorption time on the popc 
 lipid bilayer in either 10 mm phosphate buffer or pbs buffer was less 
 than 200 s ( see the time - dependent sfg signals detected in the ch 
 and oh stretching frequency range in the supporting 
 information ) . for the second step , we substituted the peptide 
 solution subphase with potassium phosphate buffer ( 6 ml in 
 total ) to wash off loosely associated peptides and recorded the sfg 
 signal in the water o 
h stretching frequency range again . for 
 the last step , we substituted the phosphate buffer subphase with a 
 solution of buffer containing 40% tfe and again collected sfg spectra 
 in the water o 
h stretching frequency range and the amide i 
 frequency range . for the c - terminal peptide
pip2 experiments 
 were performed in the same way as popc experiments except that when 
 making bilayers , lipids with a 9:1 molar ratio of popc : pip2 were used . because peptides were used at the same concentration 
 in these experiments yet likely have different affinities and because 
 water signals are also strongly affected by net charge as well as 
 charge distribution in the peptides ( and other effects ) , we defined 
 peptides as weakly membrane associated if the water signal recovered 
 after the buffer wash , as opposed to direct comparison of changes 
 in the sfg signals from water o 
atr - ftir experiments 
 were performed 
 on a nicolet magna 550 ftir spectrometer .
ri , u.k . ) using the vesicle fusion 
 method . 1 ml of popc toluene solution ( 5 mg / ml ) was dried with nitrogen 
 flow and then in vacuum for 2 h. the popc power was then dissolved 
 in 10 mm phosphate d2o buffer ph 7.4 , and the mixture was 
 vortexed for 5 min before addition to the surface of the detachable 
 znse crystal to form bilayers .
after 30 min , the vesicles floating 
 in the subphase were washed away using excess buffer .
grk5 peptides 
 were then injected into the subphase ( 1.6 ml ) to achieve a concentration 
 of 11.4 m .
after the system reached equilibrium , spectra before 
 and after extensive wash with d2o buffer were recorded . 
 for grk5546565 , s- and p - polarized spectra
were 
 taken so that data analysis on the peptide orientation could be performed . 

in the camca experiments , after peptides were associated 
 with the lipid bilayers equimolar cam ( 11.4 m ) and 50 m 
 cacl2 solution were added to the subphase .
we first investigated 
 molecular interactions between the grk5 n - terminal peptides grk5231 , grk5224 , and grk52531 and a popc / popc lipid bilayer .
the popc / popc bilayer is zwitterionic , 
 and the electrostatic potential across the bilayer induces the water 
 dipoles to orient near the bilayer surface .
the water region ( detected between 2700 and 3700 cm ) monitored by sfg spectroscopy can be used 
 to determine the binding properties of ions or peptides . in our experiments , we 
 observed two broad water o 
h stretching peaks centered at 3200 
 and 3400 cm in the sfg spectrum from the lipid 
 bilayer / potassium phosphate buffer interface ( figure 2a ) .
peptides were then added into the subphase , and the system 
 was allowed to reach equilibrium .
the water oh stretching signal decreased 
 upon addition of the grk5231 or grk52531 peptides to the subphase , consistent with their interaction with 
 the popc / popc bilayers ( figures 2a and 3a ) .
sfg spectra were also collected after extensive 
 washing , but no substantial changes were observed , suggesting that 
 both grk5231 and grk52531 peptides 
 are strongly associated with the bilayer .
h 
 stretching signal only decreased slightly after the addition of the 
 grk5224 peptide to the subphase , and the sfg water 
 signal recovered after washing the interface with buffer ( figure 3c ) , consistent with grk5224 only 
 being weakly associated with the popc / popc bilayer .
thus , the highly 
 charged residues spanning residues 2531 are primarily responsible 
 for membrane binding in these peptides .
sfg signals from grk5231 indicate strong association 
 with model membranes and helical character in a more hydrophobic environment . 

( a ) spectra in the c  h and o  h stretching frequency 
 region detected from the interface between the popc / popc bilayer and 
 buffer alone ( black ) , grk5231 in 10 mm phosphate 
 buffer ph 7.4 ( red ) , after washing ( blue ) , and in a mixture of 60% 
 buffer/40% tfe ( dark cyan ) .
( b ) sfg spectra of grk5231 associated with a popc / popc bilayer in contact with peptide solution 
 in 60% 10 mm phosphate buffer
( c ) sfg spectra of grk5231 associated with 
 a popc / popc bilayer in contact with 60% pbs/40% tfe .
sfg ppp signals detected from the grk52531 and 
 grk5224 peptides indicate that the latter peptide 
 only weakly associates with model membranes .
h stretching frequency region from the interface between 
 the popc / popc bilayer and buffer alone ( black ) , grk52531 in 10 mm phosphate buffer ph 7.4 ( red ) , after washing ( blue ) , and 
 in a mixture of 60% buffer/40% tfe ( dark cyan ) .
( b ) sfg spectra in 
 the amide i frequency region from grk52531 associated 
 with a popc / popc bilayer in 60% 10 mm phosphate buffer ph 7.4/40% 
 tfe .
 h stretching frequency 
 region from the interface between the popc / popc bilayer and buffer 
 alone ( black ) , grk5224 in 10 mm phosphate buffer 
 ph 7.4 ( red ) , after washing ( blue ) , and in a mixture of 60% buffer/40% 
 tfe ( dark cyan ) .
( d ) sfg spectra in the amide i frequency region from 
 grk5224 associated with a popc / popc bilayer in 
 contact with peptide solution in 60% 10 mm phosphate buffer ph 7.4/40% 
 tfe . for all three grk5 n - terminal 
 peptides , no discernible sfg amide 

this suggests that the membrane associated peptides 
 form either ordered structures but with random orientations or essentially 
 random structures . after replacing the subphase with a 40% tfe solution , 

a strong sfg amide i signal was detected from the grk5231 peptide ( figure 2b ) , but not from grk52531 or grk5224 ( figures 3b and 3d ) , consistent with 
 only grk5231 forming -helical structure 
 when the subphase becomes more hydrophobic .
this conclusion is also 
 consistent with spectral features detected in the water o  h 
 stretching frequency range after the subphase buffer was replaced 
 by the tfe mixture .
figure 2a shows that only 
 for grk5231 , a negative peak at 3300 cm   appeared , originating from the interference between 
 the n 
h stretch signal can 
 be attributed to the backbone n  h stretch or / and the side chains 
 such as lys nh3 .
although the predicted helical propensity of grk5224 is the same as that of grk5231 , no changes in 
 the spectra upon addition of tfe were detected likely because the 
 peptide was not strongly associated with the membrane and washed off 
 in the previous step . after substituting 
 the subphase with 40% 
 tfe ,
this could 
 be interpreted as residues 218 adopting an -helical 
 conformation , consistent with a prior crystal structure of grk6 and secondary structure predictions .
this phenomenon 
 also highlights that sfg , as a second - order nonlinear spectroscopy , 
 is much more sensitive to ordered structure ( such as -helices ) 
 than disordered molecules ( such as random coils ) , which is not the 
 case for linear vibrational spectroscopy such as atr - ftir .
sfg 
 spectra collected from amide i modes of peptides and proteins using 
 different polarization combinations can be used to determine membrane 
 orientations of peptides and proteins , as shown in a previous publication . using the measured signal strength ratio of 
 the -helical contribution in the ppp and ssp spectra , we deduced 
 that in 40% tfe , the orientation angle of the helical segment ( presumed 
 to be residues 218 ) of grk5231 is 46 
  1 relative to the membrane surface normal ( with ppp/ssp = 2.08  0.01 ) if we assume 
 the peptides adopt a single orientation distribution .
interestingly , 
 this orientation angle increases to 78  11 ( with 
 ppp/ssp = 2.43  0.06 ) when 
 the ionic strength of the subphase is increased by use of pbs instead 
 of phosphate buffer ( figure 2c ) .
this result suggests that the increase in ionic 
 strength does not change the conformation of the grk5231 but rather changes the charge distribution on the peptide surface 
 and thus facilitates the interaction of helical elements of the peptide 
 with the lipid head groups .
the sfg spectrum 
 of grk5546565 ( figure 4 ) 
 is similar to that of grk5231 , in that the two 
 broad peaks at 3200 and 3400 cm decreased and 
 remained so even after extensive washing , indicating strong interaction 
 of grk5546565 with the lipid bilayer .
these two peaks could be attributed to amino acid 
 side chains , disruption of the lipid 
 bilayer , or both .
the sfg amide i spectra 
 of grk5546565 , however , is very different from 
 those of the n - terminal peptides . without changing the subphase into 
 40% tfe
the 
 peak at 1655 cm is attributed to -helical 
 structure and the shoulder at 1600 cm is 
 likely from amide groups of side chains .
the peak at 1720 cm is from carbonyl groups 
 in the disrupted lipid bilayer .
this agrees with the ch stretching 
 signal change mentioned above , supporting the hypothesis that the 
 lipid bilayer is disrupted . because the intensity is not as high as 
 that of grk5231 in 40% tfe with 10 mm phosphate 
 buffer , no discernible nh peak ( 3300 cm ) in the water range ( 30004000 cm ) was 
 detected .
orientation analysis was not performed here due to the low 
 signal - to - noise ratio of the sfg spectra and because there are multiple 
 contributions to the spectra . in summary ,
the main difference between 
 grk5546565 and grk5231 is that 
 the former is partially -helical when associated with lipid 
 bilayers without need for tfe to induce helical structure .
sfg ppp signals 
 detected from grk5546565 indicate 
 strong binding to model membranes and helical character .
( a ) sfg spectra 
 in the c  h and o  h stretching frequency region from 
 the interface between the popc / popc bilayer and buffer alone ( black ) 
 and grk5546565 associated in 10 mm phosphate buffer 
 ph 7.4 ( red ) .
( b ) sfg spectra in the amide i frequency region from 
 grk5546565 associated with a popc / popc bilayer 
 in 10 mm phosphate buffer ph 7.4 .
pip2 is known to enhance the grk5-mediated phosphorylation of 
 gpcrs . in order to test
whether this 
 enhancement is related to the membrane binding of the peptides we 
 are studying herein , we constructed ( 9:1 ) popc : pip2 lipid 
 bilayers and studied its interaction with grk5231 , grk5224 , and grk5546565 . 

these results ( see supporting information figure s2 ) were then compared to those obtained when using a pure 
 popc lipid bilayer .
the sfg intensities and signal strength ratios 
 of the amide i signals detected in the amide i frequency range using 
 different polarization combinations of the grk5231 associated with the two types of bilayers exposed to the solution 
 with 40% tfe were observed to be similar , indicating that pip2 did not enhance the adsorption of grk5231 to the lipid bilayer .
the interactions of grk5224 and grk5546565 with ( 9:1 ) popc : pip2 bilayers were also similar to those with the pure popc system .
this 
 is reminiscent of protein marcks : neither the native protein nor a 
 peptide representing its positive charged cluster requires pip2 for binding to the membrane .
however , pip2 is 
 laterally sequestered in the presence of marcks and the peptide . how pip2 can increase the autophosphorylation 
 of grk5 and phosphorylation of activated gpcrs calls for further investigation
however , it should be noted that residues 2431 are well ordered 
 in both available crystal structures of grk6 and 
 that formation of a high affinity site for pip2 may require 
 the assumption of tertiary structure by this polypeptide , as mandated 
 by the fold of the enzyme .
the study on the effect of pip2 suggests that the conclusions on peptide affinity drawn from figure 24 do not require the existence of pip2 . because sfg is sensitive to ordered 
 structures , the signals generated from ordered -helices are 
 normally much stronger than those detected from random coil . on the 
 other hand ,
atr - ftir spectroscopy detects amide i ( 16001700 
 cm ) signals with similar sensitivities from different 
 secondary structural motifs , such as -helices , random coils , 
 and -sheets from peptides and proteins .
we used atr - ftir spectra to confirm the peptide adsorption 
 behavior detected by sfg . for the atr - ftir experiments , the concentrations 
 of all peptides used were 11.4 m in 10 mm phosphate d2o buffer ( pd 7.1 ) .
for all the n - terminal peptides , the amide i peak 
 center is around 1642 cm ( figure 5 ) , indicating that the peptides are most likely random coils . 
 for grk5231 and grk52531 ,
the 
 amide i peak intensities did not change after washing with buffer , 
 but for grk5224 the amide i signal decreased to 
 about half , again suggesting a weaker interaction between grk5224 and the lipid bilayer , as suggested by the sfg 
 studies .
the reason that membrane associated grk5224 did not completely disappear after washing , as observed in sfg , 
 is likely because the peptide concentration is 3 times higher than 
 that used in sfg measurements .
atr - ftir spectra of grk5 n - terminal peptides 
 confirm weak binding 
 of grk5224 .
spectra of ( a ) grk5231 , ( b ) grk5224 , and ( c ) gkr52531 associated with a popc / popc lipid bilayer in the presence of 10 
 mm phosphate buffer ph 7.4 before ( black ) and after ( red ) buffer wash . by taking secondary derivatives 
 of the atr - ftir spectra for grk5546565 ( figure 6 )
, we found 
 two peaks centered at 1646 and 1653 cm , respectively . 

the average band position in d2o is reported to be 1652 
 cm for -helix and 1645 cm for disordered secondary structure .
therefore , the peak centered at 1646 cm is attributed 
 by random coil and the other at 1653 cm is attributed 
 to -helices , consistent with sfg results indicating that the 
 grk5 c - terminal peptide forms an -helical structure . after 
 extensive washing , the atr - ftir signal remained , suggesting a strong 
 interaction with the lipid bilayer , also compatible with the sfg data .
atr - ftir 
 spectra of grk5546565 associated with 
 a popc / popc lipid bilayer in contact with 10 mm phosphate buffer ph 
 7.4 .
atr - ftir was further used 
 to investigate the molecular interactions of grk5231 with calmodulin .
as shown in figure 7a , the addition of 
 equimolar amounts of camca and grk5231 to the subphase decreased the atr - ftir amide i signal by about 50% . 
 further extensive washing with buffer led to a more substantial decline 
 of the random coil atr - ftir signal .
however , camca could 
 not extract grk52531 from the membrane ( supporting information ) , suggesting that the 
 helix formed by residues 224 is important for high affinity 
 binding to camca .
camca also was able to extract grk5546565 from 
 our model membranes ( figure 7b ) .
the initial 
 increase in the signal after addition of camca to 
 grk5546565 was unexpected .
however , this may simply 
 reflect that when camca forms a complex with this 
 peptide , it remains associated with the membrane to a greater extent 
 than when in complex with the grk5231 peptide . 

notably , in either case , the subsequent buffer wash eliminates binding , 
 indicating weak binding .
atr - ftir signals detected before and after the 
 addition of equimolar camca to the subphase for 
 peptides ( a ) grk5231 and ( b ) grk5546565 .
the spectra correspond to before ( black ) , and after ( red ) washing , 
 to the addition of camca to the subphase ( blue ) , 
 and after subsequent washing ( dark cyan ) .
our study is a clear example of how sfg 
 and atr - ftir spectroscopies 
 complement each other as methods for interrogating the structure of 
 proteins / peptides at membrane surfaces .
because sfg is a second - order 
 nonlinear optical technique , under the electric dipole approximation , 
 it only detects signal where inversion symmetry is broken .
thus , sfg 
 can minimize the interfering effects of proteins in bulk solution . 

for example , in our studies , we measured well - defined amide signals 
 using sfg from the grk5231 peptide associated with 
 lipid bilayers in contact with solutions with 40% tfe , which generates 
 a high background signal in atr - ftir spectroscopy ( supporting information ) .
another advantage of sfg is that 
 measurements do not require d2o , which is used in atr - ftir 
 to minimize interference by h2o absorption at 1650 
 cm .
we observed a drastic change of the 
 amide i signal of grk5231 when its subphase was 
 altered to contain 40% tfe .
this change was more subtle in the atr - ftir 
 spectra because random coils and -helices have peak centers 
 close to each other ( 1647 and 1653 cm , respectively ) and usually make similar contributions to the spectra . 
 for large proteins ( e.g. , grk5 ) ,
sometimes the switch from the active 
 state to the inactive state is accompanied by conformational changes . 

the unique ability of sfg to distinguish random coils from -helices 
 might shed light on the mechanisms of these processes , which may not 
 be easily distinguishable using atr - ftir spectra . on the other hand , 
 atr - ftir can directly monitor the adsorption of unstructured peptides 
 and proteins simply by inspecting the amide i signals . because unstructured 
 domains ( e.g. , random coils ) can not be readily detected by sfg spectroscopy
, 
 the adsorption of such molecules can not be directly assessed using 
 the sfg amide i signal .
however , this goal can be achieved indirectly 
 by monitoring the ordered water signal change in sfg spectra .
errors represent standard deviations 
 of four replicates obtained in each of two individual experiments . in this work we combined sfg 
 and atr - ftir spectroscopies to study 
 the in situ membrane binding potential of two regions 
 of grk5 previously implicated in binding to phospholipid bilayers . 

the uniform orientation of water molecules near the bilayer surface 
 was exploited first , as the disappearance of the sfg water signal 
 suggests their displacement by peptide molecules .
whether or not the 
 water signal would resume after washing the system with buffer was 
 used to determine if the peptide molecules are weakly or strongly 
 adsorbed .
it was shown that of the three n - terminal peptides , only 
 grk5224 binds weakly to the lipid bilayer , suggesting 
 that grk5224 alone does not play a significant 
 role in grk5 membrane binding and that residues 2531 of the 
 grk5231 peptide , which are exceptionally basic 
 and include a tryptophan residue , are primarily responsible for membrane 
 binding in this region .
this conclusion is also supported by monitoring 
 the changes in the amide i signal from the peptides before and after 
 washing with buffer using atr - ftir . from the amide
i sfg signals we 
 found that the segment containing amino acid residues 224 
 of peptide grk5231 undergoes a conformational change 
 from a random coil into a well - ordered -helix when the hydrophobicity 
 of the environment increases ( in our experiment by substituting the 
 buffer subphase with a solution containing 40% tfe ) .
it is possible 
 that tfe emulates what happens when this region encounters either 
 an activated gpcr or its own activated kinase domain .
furthermore , 
 the interaction of this region , or of an adjacent region ( i.e. , residues 
 2531 ) , with membranes is not enough to induce order in this 
 segment .
our results are consistent with those reported previously 
 that residues 218 play an important role in protein  protein 
 interactions , such as those with activated gpcrs or with the catalytic 
 core of the enzyme to stabilize a more active state .
polarization - dependent sfg measurements were used to determine the 
 angle of the helical segment of this peptide to the surface normal . 

this angle was found to increase substantially upon an increase in 
 ionic strength of the surrounding buffer solution . with a similar 
 approach
, both sfg and atr - ftir results showed that grk5546565 was partially helical on popc lipid bilayers , even in the absence 
 of a helix - inducing agent such as tfe .
a model summarizing the membrane 
 interactions of the peptides is shown in figure 8 .
schematic showing proposed membrane interaction mechanisms of the 
 grk5 n - terminal peptide grk5231 and the grk5 c - terminal 
 peptide grk5546565 .
therefore , both n- and c - terminal peptide segments of grk5 
 contribute 
 to bilayer binding and likely account for the constitutive localization 
 of grk5 on cell membranes , even though it lacks the palmitoylation 
 found in the closely related enzymes grk4 and grk6 . both residues 
 2531 and 546565 bind strongly to membranes , as evidenced 
 by their persistence even after exhaustive washing .
however , residues 
 224 at the extreme n - terminus do not represent a strong membrane 
 binding determinant . instead ,
our results are most consistent with 
 this highly conserved region only becoming ordered when it forms protein 
protein 
 interfaces , such as when in complex with an activated gpcr or when 
 it interacts with the small lobe of the grk kinase domain .
unexpectedly , 
 pip2 does not affect the binding properties of the peptides 
 we studied .
it is possible that the n - terminal peptide does not fully 
 recapitulate the binding site for this lipid because the peptide is 
 unstructured when bound to membranes , as opposed to the analogous 
 peptide in the context of the full - length enzyme , where its structure 
 is imposed by the fold of the enzyme .
the membrane interaction mechanisms 
 of the n - terminal and c - terminal peptides are , however , different . 

previous biochemical studies showed that the c - terminal peptide likely 
 forms an amphipathic helix that enhances grk5 membrane binding .
the analogous c - terminal region has only been 
 observed in one structure of grk6 in a relatively active state , wherein it forms the expected amphipathic helix
. 
 however , the hydrophobic residues bind to the catalytic core of the 
 enzyme , and the helix seems too far from the predicted membrane surface 
 to directly engage lipids .
as phospholipids are not present in this 
 crystal structure , the c - terminal structure could represent a crystallographic 
 artifact .
alternatively , because the interactions of the c - terminus 
 of grk6 with the core of the enzyme are extensive ( buried accessible 
 surface area of 2400  ) , the packing of this helix 
 could represent the situation when grk6 is in a cytoplasmic and/or 
 autoinhibited state . because pip2 is believed to bind to 
 the 2531 region in the n - terminal region and this site is 
 a structured part of the catalytic core in prior crystal structures
, 
 this interaction may be more important for achieving proper orientation 
 of the enzyme at the membrane , whereas the c - terminal amphipathic 
 helix , which is connected to the rest of the enzyme by a 21-amino 
 acid linker , is merely important for maintaining its association at 
 the membrane .
camca is able to dissociate grk5231 and grk5546565 peptides from 
 the membrane , consistent with the ability of this protein to drive 
 grk5 off the membrane of cells and consequently to the nucleus , where 
 it is believed to phosphorylate transcription factors controlling 
 hypertrophic genes .
 g protein - coupled receptor kinase 
 5 ( grk5 ) is thought to associate 
 with membranes in part via n- and c - terminal segments that are typically 
 disordered in available high - resolution crystal structures . 
 herein 
 we investigate the interactions of these regions with model cell membrane 
 using combined sum frequency generation ( sfg ) vibrational spectroscopy 
 and attenuated total reflectance  
 fourier transform infrared 
 ( atr - ftir ) spectroscopy . 
 it was found that both regions associate 
 with popc lipid bilayers but adopt different structures when doing 
 so : grk5 residues 231 ( grk5231 ) was in 
 random coil whereas grk5546565 was partially helical . 
 
 when the subphase for the grk5231 peptide was changed 
 to 40% tfe/60% 10 mm phosphate ph 7.4 buffer , a large change in the 
 sfg amide i signal indicated that grk5231 became 
 partially helical . by inspecting the membrane behavior of two different 
 segments of grk5231 , namely , grk5224 and grk52531 , we found that residues 2531 
 are responsible for membrane binding , whereas the helical character 
 is imparted by residues 224 . 
 with sfg , we deduced that the 
 orientation angle of the helical segment of grk5231 is 46  1 relative to the surface normal in 40% tfe/60% 
 10 mm phosphate ph = 7.4 buffer but increases to 78  11 
 with higher ionic strength . 
 we also investigated the effect of pip2 in the model membrane and concluded that the popc : pip2 ( 9:1 ) lipid bilayer did not change the behavior of either 
 peptide compared to a pure popc lipid bilayer . 
 with atr - ftir , we also 
 found that ca2+calmodulin is able to extract both 
 peptides from the popc lipid bilayer , consistent with the role of 
 this protein in disrupting grk5 interactions with the plasma membrane 
 in cells . 
Introduction
Experimental
Section
Results
Discussion