1,2-dioleoyl - sn - glycero-3-phosphatidylethanolamine ( dope ) , 1,2-dioleoyl - sn - glycero-3-phospho - glycerol ( dopg ) , 1,2-dioleoyl - sn - glycero-3-phosphocholine ( dopc ) , and 1,2-dioleoyl - sn - glycero-3-phosphatidic acid ( dopa ) were purchased from avanti polar lipids . for clarity , the names of these lipids are abbreviated to pe , pg , pc , and pa , respectively . n - kcsa ( lacking first 18 residues ) proteins were expressed with an n - terminal his - tag from pt7-kcsa in escherichia coli strain bl21 and purified according to the method described previously ( raja et al . polyacrylamide gel electrophoresis ( sds - page ) , and the protein concentration was determined by using standards of bovine serum albumin . 1985 ) and proteins were reconstituted in pe : pg ( 7:3  mol% ) or pc : pa ( 7:3  mol% ) lipid mixtures with a 1:1000 protein : lipid molar ratio , and the resultant proteoliposomes were obtained as described previously ( raja et al . 2007 ) . to control the equal amounts of proteins in all experiments the amount of protein reconstituted into the proteoliposomes was determined by coomasie blue staining using standards of bovine serum albumin . small aliquots of proteoliposomes were incubated with variable concentrations of tfe for 1  h at room temperature . hcl [ ph 6.8 ] , 50% glycerol , 0.01% bromophenol blue and 10% sds ) and run on 15% acrylamide gel in the presence of 0.1% sds . silver nitrate - stained gels were scanned by a densitometer ( bio - rad ) and quantified with the program quantity one ( bio - rad ) . for stability assay , the amount of tetramer ( % ) was calculated according to the procedure described previously ( raja et al . significance ( p  <  0.05 ) was calculated using student s t - test unless otherwise mentioned . fluorescence experiments were performed using a quantamaster qm-1/2005 spectrofluorometer ( photon technology , princeton , nj ) , fitted with a xenon flash lamp at room temperature and trp fluorescence spectra were collected according to the procedure described previously ( raja et al . acrylamide quenching experiments were performed to determine the accessibility of trp residues in lipid bilayers either in the presence or absence of tfe . the quenching data was analyzed by a stern  volmer equation as described previously ( raja et al . wt- and n - kcsa ( lacking first 18 residues ) proteins were expressed with an n - terminal his - tag from pt7-kcsa in escherichia coli strain bl21 and purified according to the method described previously ( raja et al . polyacrylamide gel electrophoresis ( sds - page ) , and the protein concentration was determined by using standards of bovine serum albumin . 1985 ) and proteins were reconstituted in pe : pg ( 7:3  mol% ) or pc : pa ( 7:3  mol% ) lipid mixtures with a 1:1000 protein : lipid molar ratio , and the resultant proteoliposomes were obtained as described previously ( raja et al . to control the equal amounts of proteins in all experiments the amount of protein reconstituted into the proteoliposomes was determined by coomasie blue staining using standards of bovine serum albumin . small aliquots of proteoliposomes were incubated with variable concentrations of tfe for 1  h at room temperature . samples were mixed with an electrophoresis sample buffer ( 50  mm tris  hcl [ ph 6.8 ] , 50% glycerol , 0.01% bromophenol blue and 10% sds ) and run on 15% acrylamide gel in the presence of 0.1% sds . silver nitrate - stained gels were scanned by a densitometer ( bio - rad ) and quantified with the program quantity one ( bio - rad ) . for stability assay , the amount of tetramer ( % ) was calculated according to the procedure described previously ( raja et al . significance ( p  <  0.05 ) was calculated using student s t - test unless otherwise mentioned . fluorescence experiments were performed using a quantamaster qm-1/2005 spectrofluorometer ( photon technology , princeton , nj ) , fitted with a xenon flash lamp at room temperature and trp fluorescence spectra were collected according to the procedure described previously ( raja et al . acrylamide quenching experiments were performed to determine the accessibility of trp residues in lipid bilayers either in the presence or absence of tfe . the quenching data was analyzed by a stern  volmer equation as described previously ( raja et al . first , the intrinsic stability of kcsa tetramer reconstituted in a pe : pg ( 7:3  mol% ) lipid bilayer was determined by conventional gel electrophoresis . figure  2a represents sds gels illustrating tetramer dissociation pattern in wt and n - kcsa as a function of tfe ( vol% ) . either in the absence or presence of tfe , the amount of wt - kcsa monomer is relatively higher than n - kcsa monomer indicating low solubility of the monomeric n - kcsa as discussed in the previous studies ( raja 2010a , b , c ) . for wt - kcsa tetramer , which typically runs at ~68  kda , ~35 vol% tfe was required to dissociate the tetramer into its monomeric subunits ( ~18  kda ) . however , n - kcsa was completely destabilized at ~30  vol% tfe indicating once again that the n - terminus contributes to the stabilizing effect of full - length kcsa as shown in the previous studies ( raja 2010a , b , c ) . the disappearance of monomeric n - kcsa is an indicative of protein aggregation at either low or high tfe concentrations that can not be seen on the sds gel ( raja et al . 2007 ) . figure  2b demonstrates the quantification of representative sds gels shown in fig . 2a in which the amount of tetrameric fraction ( % ) at various tfe concentrations was calculated relative to the total amount of tetramer present in the absence of tfe ( 0 vol% ) . these results indicate that the n - terminus significantly stabilizes the tetramer in a pe : pg lipid bilayer similar to pc : pg bilayers ( raja 2010b).fig . 2tfe - induced tetramer dissociation of wt and n - kcsa in pe : pg ( 7:3  mol.% ) lipid bilayers . tetrameric ( t ) and monomeric ( m ) kcsa are indicated ; and a protein marker ( in kda ) is shown on the right . the intensities of tetramer bands were assigned as a relative value of 100% observed for a tfe - untreated ( 0 vol% tfe ) sample . data points correspond to the average    sd of three experiments tfe - induced tetramer dissociation of wt and n - kcsa in pe : pg ( 7:3  mol.% ) lipid bilayers . tetrameric ( t ) and monomeric ( m ) kcsa are indicated ; and a protein marker ( in kda ) is shown on the right . the intensities of tetramer bands were assigned as a relative value of 100% observed for a tfe - untreated ( 0 vol% tfe ) sample . data points correspond to the average    sd of three experiments to investigate the folding properties of wt and n - kcsa in a pe : pg lipid bilayer tryptophan ( trp ) fluorescence experiments were performed . the strong sensitivity of trp fluorescence has been proven to be a powerful tool in investigating kcsa folding properties in different lipid bilayer systems ( raja 2010b ; raja et al . 2007 ) . for wt - kcsa , trp fluorescence emission spectra in the absence and presence of various concentrations of tfe ( vol% ) it is interesting to observe that 510 vol% tfe increases trp fluorescence intensity , thus demonstrating the shielding effect on trp residues from the local environment . further increasing tfe concentration ( 2030 vol% ) resulted in decrease in fluorescence or fluorescence quenching ( 2533% ) followed by redshifts in emission maxima indicating an increase in hydrophilicity or increased accessibility of trp residues to their local environment possibly due to protein unfolding ( raja 2010b ; raja et al . using higher tfe concentrations ( ~40  vol% ) did not change trp fluorescence parameters significantly ( not shown ) . these results indicate that kcsa tetramer is quite stable in a pe : pg bilayer and that the folding properties of kcsa are remarkably different in this lipid system compared to other artificial membrane systems studied previously ( raja et al . 2007).fig . 3effect of tfe on trp fluorescence of wt - kcsa ( a ) and n - kcsa ( b ) in pe : pg ( 7:3  mol% ) bilayers . samples were investigated in the absence or presence of varying concentrations of tfe ( vol% ) as indicatedtable  1effects of tfe on folding properties of wt and n - kcsa in a pe : pg ( 7:3  mol% ) lipid bilayersampleswavelength ( nm)ksv ( m)tfe ( vol%)0510203005102030wt3223223223233254.0    0.34.2    0.24.4    0.65    0.45.5    0.3n3193223243253257    0.47.6    0.38    0.38.6 0.48.8    0.6parameters were derived from trp fluorescence data shown in fig . volmer quenching constants were derived from the slopes of the linear regression lines from plots of f0/f  =  1  +  ksv [ q ] shown in fig . all values are the means    sd of three experiments effect of tfe on trp fluorescence of wt - kcsa ( a ) and n - kcsa ( b ) in pe : pg ( 7:3  mol% ) bilayers . samples were investigated in the absence or presence of varying concentrations of tfe ( vol% ) as indicated effects of tfe on folding properties of wt and n - kcsa in a pe : pg ( 7:3  mol% ) lipid bilayer parameters were derived from trp fluorescence data shown in fig . 3a the stern  volmer quenching constants were derived from the slopes of the linear regression lines from plots of f0/f  =  1  +  ksv [ q ] shown in fig . all values are the means    sd of three experiments qualitatively , similar results were obtained for n - kcsa ( fig . 3b , table  1 ) in a pe : pg ( 7:3 ) bilayer . again , 510 vol% tfe induced an increase in trp fluorescence , and addition of 2030 vol% tfe resulted in fluorescence quenching ( 3039% ) . however , redshifts in emission maxima were found to be more pronounced in n - kcsa as a function of tfe concentration . taken into account changes in trp fluorescence parameters in wt and n - kcsa in both types of lipid bilayers the n - terminus seems to play a role in protecting kcsa tetramer from unfolding especially at lower tfe concentrations . the increase and decrease in fluorescence as well as redshifts in emission maxima suggested that kcsa tetramer undergoes various conformational changes at low and high tfe concentrations . thus , a more direct approach of investigating protein conformational changes by acrylamide quenching experiments was used . the main benefit of using acrylamide is that it is a hydrophilic quencher of trp fluorescence and it has a quite low permeability to lipid membranes . in addition , no charge interaction takes place between the negatively charged lipid head groups ( breukink et al . the stern  volmer quenching plots of wt and n - kcsa in a pe : pg lipid bilayer either in the absence and presence of tfe were found to be linear and are shown in fig .  4a and b , respectively . the stern volmer constants ( ksv ) are compiled in table  1 . in the absence of tfe , a significant increase in quenching constant for n - kcsa compared to wt - kcsa was observed indicating that both tetramers are slightly different with regard to their membrane organization . for wt - kcsa ( fig . 4a ) , no significant change in acrylamide quenching was observed for 510 vol% tfe ; however , a slight increase in quenching was observed at 2030 vol% tfe indicating protein unfolding as also deduced by redshift in trp fluorescence spectra ( fig . interestingly , acrylamide quenching was found to be more pronounced in n - kcsa ( fig . 4b ) indicating that in the absence of n - terminus the kcsa is slightly more sensitive to tfe - induced tetramer unfolding.fig . volmer plots of trp fluorescence quenching by acrylamide in wt - kcsa ( a ) and n - kcsa ( b ) in pe : pg ( 7:3  mol% ) bilayers . samples were investigated with or without certain concentrations of tfe ( vol% ) as indicated . the slopes of the best fit linear regression lines for each data set ( ksv values ) are shown in table  1 representative stern volmer plots of trp fluorescence quenching by acrylamide in wt - kcsa ( a ) and n - kcsa ( b ) in pe : pg ( 7:3  mol% ) bilayers . samples were investigated with or without certain concentrations of tfe ( vol% ) as indicated . the slopes of the best fit linear regression lines for each data set ( ksv values ) are shown in table  1 for comparison , the values derived from the effect of tfe on trp fluorescence and acrylamide quenching experiments of wt - kcsa in a pc : pa ( 7:3 ) lipid bilayer from the previous studies ( raja et al . 2007 ) are compiled in table  2 . in the absence of tfe , trp fluorescence emission wavelengths and stern  volmer quenching constants were found to be quite similar for wt - kcsa suggesting that the tetramer adopts a similar folding pattern both in pe : pg and pc : pa lipid bilayers ( compare table  1 and table  2 ) . however , after the addition of tfe , trp fluorescence and quenching constants were found to be significantly different among both lipid bilayers indicating that in addition to h - bonding the strong ionic interaction between the negatively charged pa and positively charged residues on wt - kcsa protects tetramer from unfolding.table  2effects of tfe on folding properties of wt and n - kcsa in a pc : pa ( 7:3  mol% ) lipid bilayersamplewavelength ( nm)ksv ( m)tfe ( vol%)0510203005102030wt320320320321.5321.53    0.62.6    0.82.5    1.22.7    0.88.0    1.8n3203203203203202.2    0.72.8    14.5    0.86.5    2.37    1.6parameters were derived from trp fluorescence data shown in fig . volmer quenching constants were derived from the slopes of the linear regression lines from plots of f0/f  =  1  +  ksv [ q ] . all values are the means    sd of three experimentsthe values for wt in pc : pa ( 7:3 ) were derived from the previously published work ( raja et al . 2007)the values were derived from trp fluorescence emission spectra and acrylamide quenching plots ( data not shown ) effects of tfe on folding properties of wt and n - kcsa in a pc : pa ( 7:3  mol% ) lipid bilayer parameters were derived from trp fluorescence data shown in fig . volmer quenching constants were derived from the slopes of the linear regression lines from plots of f0/f  =  1  +  ksv [ q ] . all values are the means    sd of three experiments the values for wt in pc : pa ( 7:3 ) were derived from the previously published work ( raja et al . 2007 ) the values were derived from trp fluorescence emission spectra and acrylamide quenching plots ( data not shown ) for n - kcsa in pc : pa ( table  2 , data not shown ) , no change in trp emission wavelength was observed either in the absence or presence of tfe . however , significantly reduced accessibility to acrylamide or decrease in stern volmer quenching constant compared to n - kcsa in pe : pg was calculated ( compare table  1 and table  2 ) . these results indicate that the n - terminus significantly contributes to protect kcsa tetramer from unfolding as a result of its deep insertion into pe lipid headgroup . however , in pc : pa , the n - terminus seems to have a marginal role in protecting kcsa against tfe - induced unfolding because the positively charged region ( arg117 , arg121 or arg122 ) close to the intracellular membrane surface at the c - terminus has a preferential interact with pa as also shown in the previous studies ( raja et al . first , the n - terminus significantly increased the tetrameric stability in a pe : pg ( 7:3  mol% ) lipid bilayer which mimics the natural membrane environment for kcsa assembly in bacterial membranes . the stabilizing effect of the n - terminus was similar to what has been shown in the previous studies ( raja 2010b , c ) indicating that n - terminus indeed contributes to stabilize the tetrameric structure via electrostatic interaction regardless of the size of zwitterionic pe or pc headgroups . an increase in trp fluorescence at low concentrations of tfe was unique for pe : pg lipid bilayer system , because fluorescence quenching was observed for other membrane systems ( raja et al . furthermore , tfe - induced trp fluorescence quenching for pe : pg system was not as drastic as determined for pure pc or pc : pg lipid bilayers ( raja et al . in fact , trp fluorescence and acrylamide quenching parameters were found to be quite close to pc : pa lipid bilayer system ( raja et al . these observations suggest that the tetramer has a maximum stability in a pe : pg system . in other words it is important to mention here that pg , as a bilayer lipid , is required for the membrane stability of a pe : pg lipid bilayer because pe is a nonbilayer lipid and can not form bilayer structures alone . it is known that in the pe : pg bilayer pe interacts more strongly with pg than with pe , thus controlling the bacterial membrane permeability and stability ( murzyn et al . 2005 ) . such information can also be supported by the fact that in additional experiments the pe : pa ( 7:3  mol% ) lipid combination resulted in a complete loss of kcsa protein incorporation efficiency indicating that no bilayer structures were formed for a pe : pa system that could support kcsa reconstitution ( not shown ) . these observations also suggest that pe does not interact with pa because it may behave as a nonbilayer lipid as a result of its small headgroup size as pe . one possibility is that the small headgroup of pe may increase the space available for partitioning of tfe at the lipid / water interface as shown by carboxyfluorescein ( cf ) leakage experiment in the earlier studies ( van den brink - van der laan et al . another reasonable explanation is that pe : pg bilayer system is more compact and resistant to tfe - induced membrane destabilization as a result of the preferential interaction of pe with pg , as discussed above . in case of a pc : pa lipid bilayer , no significant difference has been observed in the cf leakage pattern compared to pc or pc : pg lipid bilayers ( raja et al . pe favors the deep incorporation of proteins because of its smaller headgroup size ( dowhan and bogdanov 2009 ; heller et al . pe molecules are known to restrict the dissipation of clustered phospholipids especially anionic phospholipids ( bazzi and nelsestuen 1992 ) . a similar phenomenon can be assumed by which pe may induce lateral clustering of anionic pg in a pe : pg lipid bilayer that allows pg cluster to surround kcsa via electrostatic interactions involving arg64 and arg89 at the extracellular surface , arg11 , lys14 , or arg19 at the n - terminus , and arg117 , arg121 , or arg122 close to the intracellular membrane surface . the functional and structural importance of these residues can be explained by the fact that deletion of these arg residues as hot spots , particularly at the c - terminus , impairs kcsa assembly in a heterologous e. coli expression system which contains pe and pg lipids in the inner membrane ( molina et al . thus , the extreme stability and tight packing of kcsa will be followed by binding of kcsa positively charged residues ( arg and/or lys ) to negatively charged pg and then the deep insertion of membrane - binding domains would take place by extensive hydrophobic interaction and h - bonding between pe and kcsa , which is not possible for pc . the hypothetical models were derived to compare kcsa folding in pc , pe , or pa containing lipid bilayers ( fig . , kcsa tetramer adopts a wobbly or loosely packed structure , whereas in bilayers containing either pe or pa the tetramer has a more compact structure as a result of the deep insertion of the lipid binding domains of kcsa.fig . 5a hypothetical scheme of kcsa tetramer folding in pc ( a ) or pe ( b ) containing lipid bilayers . in pc ( large headgroup ) however , in the presence of pe ( small headgroup ) , the tetramer is squeezed and adopts a more compact structure similar to pa - kcsa complex ( pe    pa ) . the dotted lines represent a hypothetical scale to elaborate tetramer stretching or squeezing pattern in pc or pe    pa lipid bilayers , respectively . the n- and c - termini are not shown a hypothetical scheme of kcsa tetramer folding in pc ( a ) or pe ( b ) containing lipid bilayers . in pc ( large headgroup ) , the tetramer is loosely packed . however , in the presence of pe ( small headgroup ) , the tetramer is squeezed and adopts a more compact structure similar to pa - kcsa complex ( pe    pa ) . the dotted lines represent a hypothetical scale to elaborate tetramer stretching or squeezing pattern in pc or pe    pa lipid bilayers , respectively . the n- and c - termini are not shown in this context , the lipid headgroup properties of pe can be taken into further consideration . pe behaves as a cone - shaped lipid similar to the molecular shape of anionic pa ( kooijman et al . 2007 ) . pe is known to facilitate membrane association and insertion of monomeric kcsa because of its small headgroup size ( ridder et al . similarly , the small size of the pa headgroup as well as the negative charge allow stronger interaction and tight docking of the kcsa subunit into the membrane , thus increasing channel stability via h - bonding / electrostatic interactions ( raja et al . it is fascinating that both zwitterionic pe and anionic pa are completely different in their net charge . however , the folding and stabilizing effects of pe and pa on kcsa structure seem related , which might be due to the similar small headgroup size of both lipids . a more in depth study varying the fatty acid composition of pe however , the fatty acid compositions of pe and pc have been shown to have differing effects on other membrane proteins ( bogdanov et al . 1999 , 2010 ) , which are not yet completely understood .
phospholipid headgroups act as major determinants in proper folding of oligomeric membrane proteins . 
 the k+-channel kcsa is the most popular model protein among these complexes . 
 the presence of zwitterionic nonbilayer lipid phosphatidylethanolamine ( pe ) is crucial for efficient tetramerization and stabilization of kcsa in a lipid bilayer . in this study , 
 the influence of pe on kcsa folding properties was analyzed by tryptophan fluorescence and acrylamide quenching experiments and compared with the effect of anionic phosphatidic acid ( pa ) . 
 the preliminary studies suggest that the small size and hydrogen bonding capability of the pe headgroup influences kcsa folding via a mechanism quite similar to that observed for anionic pa .