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  Importance of indole N \ H hydrogen bonding in the organization anddynamics of gramicidin channels Arunima Chaudhuri a , Sourav Haldar a,1 , Haiyan Sun b , Roger E. Koeppe II  b , Amitabha Chattopadhyay a, ⁎ a CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India b Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA a b s t r a c ta r t i c l e i n f o  Article history: Received 12 August 2013Received in revised form 10 October 2013Accepted 14 October 2013Available online 19 October 2013 Keywords: GramicidinIndole hydrogen bonding1-MethylindoleIon channelREESMembrane penetration depth The linear ion channel peptide gramicidinrepresents anexcellent model for exploring theprinciples underlyingmembrane protein structure and function, especially with respect to tryptophan residues. The tryptophanresidues in gramicidin channels are crucial for the structure and function of the channel. In order to test theimportance of indole hydrogen bonding for the biophysical properties of gramicidin channels, we monitoredthe effect of N-methylation of gramicidin tryptophans, using a combination of steady state and time-resolved 󿬂 uorescence approaches along with circular dichroism spectroscopy. We show here that in the absence of thehydrogen bonding ability of tryptophans, tetramethyltryptophan gramicidin (TM-gramicidin) is unable tomaintain the single stranded, head-to-head dimeric channel conformation in membranes. Our results showthat TM-gramicidin displays a red-shifted  󿬂 uorescence emission maximum, lower red edge excitation shift(REES), and higher  󿬂 uorescence intensity and lifetime, consistent with its nonchannel conformation. This is inagreement with the measured location (average depth) of the 1-methyltryptophans in TM-gramicidin usingthe parallax method. These results bring out the usefulness of 1-methyltryptophan as a  󿬂 uorescent tool toexamine the hydrogen bonding ability of tryptophans in proteins and peptides. We conclude that changes inthe hydrogen bonding ability of tryptophans, along with coupled changes in peptide backbone structure inducethe loss of single stranded  β 6.3 helical dimer conformation. These results agree with earlier results from size-exclusion chromatography and single-channel measurements for TM-gramicidin, and con 󿬁 rm the importanceof indole hydrogen bonding for the conformation and function of ion channels and membrane proteins.© 2013 Elsevier B.V. All rights reserved. 1. Introduction Biological membranes represent complex two-dimensional, non-covalent assemblies of a diverse variety of lipids and proteins. Theyprovide an identity to the cell and facilitate cellular communicationand information processing. Membrane proteins are workhorses of the cellular machinery. About 30% of all proteins are predicted to bemembrane proteins and ~50% of all proteins are membrane proteinsfor eukaryotic cells [1,2]. The crystallization efforts of membraneproteins in their native conditions are often complicated, and poseconsiderable challenge due to the intrinsic dependence of membraneprotein structure on surrounding membrane lipids [3]. Approaches based on NMR and  󿬂 uorescence spectroscopy have proved useful inelucidating the organization, topology and orientation of membraneproteins and peptides [4,5]. An additional advantage of spectroscopicapproaches is that the information obtained is dynamic in nature,necessary for understanding membrane protein function.Transmembrane proteins and peptides have characteristicstretches of amino acids capable of interacting with the membranebilayer and are reported to have a signi 󿬁 cantly higher tryptophancontent than soluble proteins [6]. Tryptophan residues are believed to be crucial in the structure and function of membrane proteinsand peptides [7 – 12]. A major observation is that tryptophans inmembrane proteins and peptides are not uniformly distributed, buttend to be localized toward the membrane interface. Interestingly,the interfacial region in membranes is characterized by uniquemotional and dielectric properties, distinct from both the bulkaqueous phase and the hydrocarbon-like interior of the membrane[12,13]. A unique feature of tryptophan is its ability to participatein both hydrophobic and polar interactions. Among the naturallyoccurring amino acids, tryptophan shows the highest tendency tolocalize at the interface, based on partitioning of model peptides tomembrane interfaces. Besides aromaticity and ring shape, hydrogen Biochimica et Biophysica Acta 1838 (2014) 419 – 428  Abbreviations: TM-gramicidin,tetramethyltryptophangramicidin;POPC,1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine; DMPC, 1,2-dimyristoyl- sn -glycero-3-phosphocholine;DOPC, 1,2-dioleoyl- sn -glycero-3-phosphocholine; 5-PC, 1-palmitoyl-2-(5-doxyl)stearoyl- sn -glycero-3-phosphocholine; 12-PC, 1-palmitoyl-2-(12-doxyl)stearoyl- sn -glycero-3-phosphocholine; 2-AS, 2-(9-anthroyloxy)stearic acid; 12-AS, 12-(9-anthroyloxy)stearic acid; REES, red edge excitation shift; SUV, small unilamellarvesicles; CD, circular dichroism; LED, light emitting diode ⁎  Corresponding author. Tel.: +91 40 2719 2578; fax: +91 40 2716 0311. E-mail address:  amit@ccmb.res.in (A. Chattopadhyay). 1 Present address: Room10D14,10 CenterDrive, National Institute of Child Health andHuman Development, National Institutes of Health, Bethesda, MD, USA.0005-2736/$  –  see front matter © 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.bbamem.2013.10.011 Contents lists available at ScienceDirect Biochimica et Biophysica Acta  journal homepage: www.elsevier.com/locate/bbamem  bonding could play a role in the partitioning of the indole ring[8,14,15]. The overall role of tryptophan residues in the structureand function of membrane proteins and peptides is apparent fromthe observation that substitution or deletion of tryptophans oftenresults in reduction or loss of their function [16 – 20].Thelinearpeptidegramicidinformsprototypicalionchannelsspeci 󿬁 cfor monovalent cations and has been extensively used to explore theorganization, dynamics, and function of membrane-spanning channels[21,22]. Gramicidin is a multi-tryptophan peptide (Trp-9, 11, 13, and15; see Fig. 1a) which serves as an excellent model for transmembranechannelsduetoanumberofreasonssuchassmallsize,readyavailabilityand the relative ease with which chemical modi 󿬁 cations can beperformed. These special features make gramicidin unique among smallmembrane-active peptides and provide the basis for its use to explorethe principles that govern the folding and function of membrane-spanning channels [21 – 23]. Interestingly, gramicidin channels sharevital structural features involving ion selectivity with complex ionchannels such as KcsA potassium channels [24]. Gramicidin assumes awiderangeofenvironment-dependentconformationsduetoitsuniquesequence of alternating  L  - and  D -chirality. Two major conformationsadopted by gramicidin in various environments are: (i) the singlestranded  β 6.3 helical dimer (the  ‘ channel ’  form), and (ii) the doublestranded intertwined helix (collectively known as the  ‘ nonchannel ’ form) [22]. The amino terminal-to-amino terminal single-stranded β 6.3 helical dimer form is the thermodynamically preferred confor-mation in membranes and membrane-mimetic media. In this confor-mation, the tryptophan residues remain clustered at the membrane – water interface [25 – 28]. Interestingly, the membrane interfaciallocalization of tryptophan residues is absent in  ‘ nonchannel ’  confor-mations and the tryptophan residues are distributed along themembrane axis [10,22,25]. Nonchannel conformations have beenshown to exist in membranes with polyunsaturated lipids [29], and inmembranes with increased acyl chain lengths under hydrophobicmismatch conditions [30,31]. a  HCOVGALAVVV W L W L W L W NHCH 2 CH 2 OH 1513119 Gramicidin c 1-Methylindole 24567 NH 24567 Indole b INTERFACEINTERFACEHYDROCARBON CORE CH 2 CH 2 CCN + OPO – OOOOOO      C     H     2      C     H       2  C     C     N     +      O P     O     –      O     O     O     O     O     O   CH 2 CH 2 CCN + OPO – OOOOOO      C     H     2        C     H     2  C     C     N     +      O P     O     –      O     O     O     O     O     O Nonchannel(tryptophans distributed)Channel(tryptophans clustered)      C     H     2        C     H     2  C     C     N     +      O P     O     –      O     O     O     O     O     O   CH 2 CH 2 CCN + OPO – OOOOOO Fig.1. (a)AminoacidsequenceofgramicidinAhighlightingthepositionsofthefourtryptophans.Alternating D -aminoacidresiduesareunderlined.Intheanalog(TM-gramicidin)usedinthis study, four tryptophan residues in gramicidin are replaced by 1-methyltryptophan residues. (b) A schematic representation of the nonchannel and channel conformations of gramicidinshowingthelocalizationoftryptophanresiduesinthemembranebilayer.Tryptophansareclusteredtowardthemembraneinterfaceinthechannelconformation.Incontrast,tryptophansaredistributedalongthebilayernormalinthenonchannelconformation.Seetextforotherdetails.Adaptedandmodi 󿬁 edfromRef.[12].(c)Chemicalstructuresofindoleand 1-methylindole.Themajordifferencebetweenindoleand1-methylindoleisintheirabilitytoformhydrogenbonds.Whileindolecanformhydrogenbondwithits \ NHgroup,thisabilityislostin1-methylindole.Interestingly,thedipolemomentsofindoleand1-methylindole(shownasavector)aresimilarindirectionandmagnitude.Seetextforotherdetails.Adaptedandmodi 󿬁 ed from Ref. [43].420  A. Chaudhuri et al. / Biochimica et Biophysica Acta 1838 (2014) 419 – 428  Gramicidinhasprovedtobeapowerfulmodelforelucidatingtheroleof tryptophan at the membrane – water interface for maintaining ionchannel structure and assembly [16,17,32 – 37]. The tryptophan residuesin gramicidin channels have previously been shown to be crucial for thestructure and function of the ion channel [16,17,32 – 35]. The importanceof tryptophans is apparent from previous observations that the cationconductivity of the channel is reduced upon substitution of one or all of the tryptophan residues by phenylalanine, tyrosine or naphthylalanine[16,17,32],andalsouponultravioletirradiationorchemicalmodi 󿬁 cationof the tryptophans [35,38,39]. It has been previously shown thatgramicidins with Trp → Phe or Tyr substitutions have greater dif  󿬁 cultyinformingmembrane-spanningdimericchannels[16,35].Unfortunately,these results do not provide information on the speci 󿬁 c properties of tryptophan that contribute to the loss of channel structure and function.The loss in structure and function upon substitution of tryptophan withphenylalanine or tyrosine could be attributed to the loss of dipolemoment, lack of hydrogen bonding ability, change in hydrophobicity, oracombinationofthesefactors.Amongthese,theroleofindoleringdipolemomentwaspreviouslyexploredbyusing5- 󿬂 uorotryptophaninsteadof tryptophan[34,40].Thissubstitutionincreasestheindoledipolemomentwithout altering other properties.In thecontextof theimportanceof hydrogen bondingin membraneprotein structure and function [41,42], tryptophan residues were previously modi 󿬁 ed to 1-methyltryptophan in order to evaluate thecontribution of the hydrogen bonding ability of tryptophans inmaintaining the channel conformation of gramicidin (see Fig. 1c forthe chemical structures of indole and 1-methylindole) [43]. Suchmodi 󿬁 cation results in the loss of hydrogen bonding ability of tryptophans. Yet, properties such as aromaticity and ring shape remaininvariant. Importantly, the magnitude (~2.1D for tryptophan and 2.2Dfor 1-methyltryptophan) and direction of the dipole moment are notaltered (see Fig. 1b) [43]. In this work, we explored the membrane organization and dynamics of the N-methylated tryptophan analog of gramicidin,  i.e. , tetramethyltryptophan gramicidin (TM-gramicidin), inwhich all four tryptophans are replaced by 1-methyltryptophanresidues. We applied a combination of   󿬂 uorescence approaches whichinclude red edge excitation shift (REES) analysis,  󿬂 uorescence lifetimeand anisotropy measurements, membrane penetration depth mea-surement, and circular dichroism (CD) spectroscopy toward this goal.Our results show that in the absence of the hydrogen bonding abilityof tryptophans, TM-gramicidin is not able to maintain the singlestranded, head-to-head dimeric channel conformation in membranes.These results are consistent with recent single-channel and solid-stateNMR results using TM-gramicidin [43]. 2. Materials and methods  2.1. Materials POPC, DOPC, 5-PC and 12-PC were obtained from Avanti Polar Lipids(Alabaster, AL). 2-AS and 12-AS were from Molecular Probes (Eugene,OR). Gramicidin A ′  (from  Bacillus brevis ) and DMPC were purchasedfromSigmaChemicalCo.(St.Louis,MO).TM-gramicidinwassynthesizedasdescribedearlier[43].Concentrationsofbothpeptideswerecalculatedusing a molar extinction coef  󿬁 cient ( ε ) of 20,700M − 1 cm − 1 at 280nm.Lipids were checked for purity by thin layer chromatography on silicagel precoated plates (Sigma) in chloroform/methanol/water (65:35:5,v/v/v) and were found to give a single spot in all cases when visualizedupon charring with a solution containing cupric sulfate (10%, w/v) andphosphoric acid (8%, v/v) at 150 °C [44]. The concentration of phospholipids was determined by phosphate assay subsequent to totaldigestionbyperchloricacid[45].DMPCwasusedasaninternalstandardto assess lipid digestion. All other chemicals used were of the highestpurity available. Solvents used were of spectroscopic grade. Water waspuri 󿬁 ed through a Millipore (Bedford, MA) Milli-Q system and used forall experiments.  2.2. Methods 2.2.1. Sample preparation Experiments were performed using SUV of POPC containing 2%(mol/mol) gramicidin or TM-gramicidin. In general, 1280nmol of POPCin methanol was mixed with 25.6nmol of gramicidin or TM-gramicidinin methanol. A few drops of chloroform were added to this solution.The solution was mixed well and dried under a stream of nitrogenwhile warming gently (~40°C), and dried further under a high vacuumfor at least 3h. The dried  󿬁 lm was swelled in 1.5ml of 10mM sodiumphosphate, 150 mM sodium chloride, pH 7.2 buffer, and samples werevortexed for 3min to uniformly disperse the lipid and peptide. Sampleswere sonicated to clarity under argon (~50 min in short bursts whilebeingcooledinanice/watermixture)usingaBransonmodel250soni 󿬁 er(Branson Ultrasonics, Danbury, CT) 󿬁 tted with a microtip. The sonicatedsamples were centrifuged at 15,000rpm in a Heraeus Biofuge centrifuge(DJB Labcare, Buckinghamshire, U.K.) for 15min to remove the titaniumparticles shed from the microtip during sonication, and incubated for12 h at 65 °C with continuous shaking to convert to the channelconformation [46,47]. Samples were incubated in a dark at room temperature (~25°C) for 1h before  󿬂 uorescence or CD measurements.Background samples were prepared the same way except that thepeptide was omitted. All experiments were done with multiple sets of samples at room temperature (~25°C).  2.2.2. Circular dichroism (CD) measurements CD measurements were carried out at room temperature (~25°C)with a JASCO J-815 spectropolarimeter (Tokyo, Japan) calibrated with(+)-10-camphorsulfonicacid. Spectra were scanned in a quartz opticalcell with a path length of 0.1 cm, and recorded in 0.5nm wavelengthincrements and band width of 2 nm. For monitoring changes insecondary structure, spectra were scanned from 200 to 260nm in thefar-UV range. The scan rate was 50nm/min and each spectrum is theaverage of 8 scans with a full scale sensitivity of 100 mdeg. Spectrawere corrected for background by subtraction of appropriate blanks.Data are represented as mean residue ellipticities and calculated usingthe equation: θ ½  ¼  θ obs =  10Cl ð Þ ð 1 Þ where θ obs  istheobservedellipticityin mdeg, l isthepathlength incm,and C is the concentration of peptide bonds in mol/l.  2.2.3. Steady state  󿬂 uorescence measurements Steady state  󿬂 uorescence measurements were performed with aHitachi F-4010 spectro 󿬂 uorometer (Tokyo, Japan) using 1 cm pathlength quartz cuvettes. Excitation and emission slits with a nominalbandpass of 5 nm were used for all measurements. Backgroundintensitiesofsamplesinwhichthepeptidewasomittedweresubtractedfrom each sample spectrum to cancel out any contribution due to thesolvent Raman peak and other scattering artifacts. The spectral shiftsobtained with different sets of samples were identical in most cases, orwere within ±1 nm of the ones reported. Fluorescence anisotropymeasurements were performed at room temperature (~25°C) using aHitachi polarization accessory. Fluorescence anisotropy values werecalculated from the equation [48]:r  ¼ I VV  − GI VH I VV   þ  2GI VH ð 2 Þ where I VV   and I VH  are the measured  󿬂 uorescence intensities (afterappropriate background subtraction) with the excitation polarizervertically oriented and emission polarizer vertically and horizontallyoriented, respectively. G is the grating correction factor and is the ratioof theef  󿬁 cienciesof the detection system for vertically andhorizontallypolarizedlights,andisequaltoI HV  /I HH .Allexperimentswereperformed 421  A. Chaudhuri et al. / Biochimica et Biophysica Acta 1838 (2014) 419 – 428  with multiple sets of samples and average values of anisotropy arereported.  2.2.4. Time-resolved  󿬂 uorescence measurements Fluorescence lifetimes were calculated from time-resolved  󿬂 uo-rescence intensity decays using an IBH 5000F NanoLED equipment(Horiba Jobin Yvon, Edison, NJ) with DataStation software in the time-correlated single photon counting mode. A pulsed light emitting diode(LED) (NanoLED-17) was used as the excitation source. This LEDgenerates optical pulse at 294 nm with a pulse duration less than750ps,andisrunata1MHzrepetitionrate.TheLEDpro 󿬁 le(instrumentresponse function) was measured at the excitation wavelength usingLudox (colloidal silica) as the scatterer. In order to optimize the signaltonoiseratio,10,000photoncountswerecollectedinthepeakchannel.Allexperimentswereperformedusingemissionslitswithabandpassof 6nm or less. The sample and the scatterer were alternated after every5% acquisition to ensure compensation for any shape and timing driftsthat could occur during the period of data collection. This arrangementalso prevents any prolonged exposure of the sample to the excitationbeam, thereby avoiding any possible photodamage to the  󿬂 uorophore.Data were stored and analyzed using DAS 6.2 software (Horiba JobinYvon, Edison, NJ). Fluorescence intensity decay curves so obtainedwere deconvoluted with the instrument response function andanalyzed as a sum of exponential terms:F t ð Þ ¼ X i α i exp − t = τ i ð Þ ð 3 Þ where F(t) is the  󿬂 uorescence intensity at time t and  α i  is a pre-exponential factor representing the fractional contribution to the time-resolved decay of the component with a lifetime  τ i  such that  Σ  i α i =1.Decay parameters were recovered using a nonlinear least squaresiterative  󿬁 tting procedure based on the Marquardt algorithm [49]. Theprogram also includes statistical and plotting subroutine packages[50]. The goodness of   󿬁 t of a given set of observed data and the chosenfunction was evaluated by the  χ  2 ratio, the weighted residuals [51],and the autocorrelation function of the weighted residuals [52]. A  󿬁 twas considered acceptable when plots of the weighted residuals andthe autocorrelation function showed random deviation about zerowith a minimum χ  2 value not more than 1.4. Intensity-averaged meanlifetimes( b τ N )fortriexponentialdecaysof  󿬂 uorescencewerecalculatedfrom the decay times and pre-exponential factors using the followingequation [48]: τ h i ¼  α 1 τ 21  þ α 2 τ 22  þ α 3 τ 23 α 1 τ 1  þ α 2 τ 2  þ α 3 τ 3 :  ð 4 Þ  2.2.5. Depth measurements using the parallax method The actual spin (nitroxide) content of the spin-labeled phos-pholipids (5- and 12-PC) was assayed using  󿬂 uorescence quenching of anthroyloxy-labeled fatty acids (2- and 12-AS) as described earlier[53]. For depth measurements, SUVs were prepared by sonicationas described above (see Section 2.2.1). These samples were madewith 160 nmol of DOPC containing a 15 mol% spin-labeled phos-pholipid (5- or 12-PC) and 3.2 nmol of TM-gramicidin. Duplicatesamples were prepared in each case except for samples lacking thequencher(5-or12-PC)wheretriplicateswereprepared.Backgroundsamples lacking the peptide were prepared in all experiments, andtheir  󿬂 uorescence intensities were subtracted from the respectivesample  󿬂 uorescence intensity. 3. Results Circular dichroism spectroscopy is a convenient method to monitorconformations of gramicidin in membranes [25,36,43,54]. CD spectrumfor the channel conformation of gramicidin displays two characteristicpeaks of positive ellipticity at ~218 and 235 nm, a valley at 230 nm,and a negative ellipticity below 208 nm. These are considered to becharacteristicof thesingle-stranded β 6.3 conformation.Thenonchannelform of gramicidin exhibits a large negative peak at ~229nm, a weakerpositive peak at ~218nm, and a positive ellipticity below 208nm. Weexamined the backbone conformation of TM-gramicidin using CDspectroscopyinPOPCinordertocomplementtheCDspectrapreviouslyreported for the analog in DMPC and DOPC [43]. Fig. 2 shows the CD spectrum of TM-gramicidin. The  󿬁 gure also shows the spectrum forgramicidin in the channel conformation (induced by sonicationfollowed by prolonged heat incubation at 65 °C) as a reference.Interestingly,theCDspectrumofTM-gramicidinresemblesthespectralfeatures of the nonchannel conformation.Thenormalized 󿬂 uorescenceemissionspectraofgramicidinandTM-gramicidin in POPC vesicles are shown in Fig. 3. The  󿬁 gure shows thattryptophans in the channel form of gramicidin display an emissionmaximum 2 of 333 nm (when excited at 280 nm) in agreement withprevious results [25]. In contrast, the emission maximum of TM-gramicidindisplaysasigni 󿬁 cant redshift andisat 340nm.Interestingly,the  󿬂 uorescence of 1-methyltryptophan (the  󿬂 uorophore in TM-gramicidin) has been reported to be sensitive to its immediateenvironment [55,56]. The relatively red-shifted emission maximum of TM-gramicidin could be indicative of the average environment expe-rienced by 1-methyltryptophans in TM-gramicidin. It should be notedhere that the emission maxima of gramicidin and TM-gramicidin inmethanol do not differ appreciably (data not shown). The difference inthe emission maximum observed in a membrane-bound conditiontherefore can be attributed to the conformational differences adoptedby gramicidin and TM-gramicidin. We have previously reported a red-shifted emission maximum in the case of the nonchannel conformationof gramicidin [25].The inset in Fig. 3 shows the relative  󿬂 uorescence intensities of gramicidin and TM-gramicidin at their respective emission maximum.TM-gramicidin exhibits an appreciable increase (~3.5 fold) in  󿬂 uo-rescence intensity relative to gramicidin. This could be attributed toboth the photophysical properties of 1-methyltryptophan and theapparent nonchannel conformation of TM-gramicidin in POPCmembranes. The quantum yield of 1-methyltryptophan has beenreported to be higher than that of tryptophan [56], which couldlead to an increase in  󿬂 uorescence intensity for TM-gramicidin. Wehave earlier shown that the conformational change of gramicidinin membranes from the nonchannel to channel form is accompaniedby a reduction in  󿬂 uorescence intensity [25]. In other words, thenonchannel conformation is characterized by an increased  󿬂 uo-rescence, possibly due to the fact that there is a distribution of tryptophans along the bilayer normal in this conformation. Theincrease in  󿬂 uorescence intensity in the case of TM-gramicidin couldtherefore be due to the relatively nonpolar environment in which the 󿬂 uorophore 1-methyltryptophan of TM-gramicidin is localized, sincea reduction in polarity is associated with enhancement of   󿬂 uorescence[55]. Yet another reason could be the release of quenching in thenonchannel conformation due to absence of aromatic – aromatic(stacking) interaction between the  󿬂 uorophores at positions 9 and15 observed in the channel conformation [28,37].REES is de 󿬁 ned as the shift in the wavelength of the maximum 󿬂 uorescence emission toward higher wavelengths, caused by a shift inthe excitation wavelength toward the red edge of the absorption band.This effect assumes relevance for polar  󿬂 uorophores in a motionallyrestricted environment where the dipolar relaxation time for the 2 We have used the term maximum of   󿬂 uorescence emission in a somewhat broadersense here. In every case, we have monitored the wavelength corresponding to themaximum  󿬂 uorescence intensity, as well as the center of mass of the  󿬂 uorescenceemission, in the symmetric part of the spectrum. In most cases, both these methodsyielded thesame wavelength. Incaseswhere minordiscrepancieswerefound,thecenterof mass of emission has been reported as the 󿬂 uorescence maximum.422  A. Chaudhuri et al. / Biochimica et Biophysica Acta 1838 (2014) 419 – 428  solvent shell around a  󿬂 uorophore becomes comparable to or longerthan its  󿬂 uorescence lifetime [12,13,57 – 60]. An attractive aspect of REES is that it allows the monitoring of the mobility parameters of theenvironment itself (represented by the relaxing solvent molecules)using the  󿬂 uorophore merely as a reporter group. REES has proved tobe a useful tool to monitor gramicidin conformations in membranesand membrane-mimetic environments [25,28,36,37,61,62].Fig. 4 shows the shifts in the maxima of   󿬂 uorescence emission of gramicidin and TM-gramicidin as a function of excitation wavelength.The  󿬁 gure shows that the emission maximum of gramicidin is shiftedfrom 333 to 340nm in response to a change in excitation wavelengthfrom 280 to 307 nm. This corresponds to a REES of 7 nm. TM-gramicidin, on the other hand, exhibits a relatively modest shift from340 to 343 nm (corresponding to a REES of 3 nm), upon change inexcitation wavelength from 280 to 307 nm. Such dependence of theemission maximum on excitation wavelength is representative of REES. It is possible that there could be further red shift upon excitationbeyond 307nm. We found it dif  󿬁 cult to work in this wavelength rangeduetoalowsignaltonoiseratioandartifactsdue tothesolventRamanpeak that sometimes remained even after background subtraction.We previously reported that the magnitude of REES could becorrelatedwiththeverticallocalization(depth)ofthegiven 󿬂 uorophoreinthemembrane[63].Fluorophorespresentinthemembraneinterfacial region,characterizedbyrestricteddynamicsduetothemobilitygradientof membranes in the vertical (z) direction [64], display greater REESrelative to  󿬂 uorophores localized in the deeper (more  󿬂 uid) regions of the membrane. The inset in Fig. 4 shows that the magnitude of REES(3 nm) exhibited by TM-gramicidin is considerably less compared toREES (7 nm) of gramicidin in the channel conformation. As statedabove, a characteristic feature of the tryptophan residues in thegramicidin channel conformation is that they remain clustered at themembrane – water interface [25 – 28]. However, this is not true for thenonchannelconformationwherethetryptophanresiduesaredistributedalong the membrane axis [10,22,25]. As a result, the nonchannel conformation of gramicidin has earlier been characterized by modestREES[25].ThemagnitudeofREESexhibitedbyTM-gramicidinthereforeindicates that the localization of   󿬂 uorophores (1-methyltryptophan) inTM-gramicidin is deeper than the localization of tryptophans ingramicidin channels. This is also consistent with their lack of hydrogenbonding ability, since the membrane interface offers suitable chemistryfor hydrogen bonding [13]. These results imply a nonchannel-likeorganization for TM-gramicidin in the membrane. This is in agreementwith the results from CD measurements (see Fig. 2).Fluorescence lifetime serves as a sensitive indicator of the localenvironment and polarity in which a given  󿬂 uorophore is localized[65]. A typical decay pro 󿬁 le of TM-gramicidin in POPC vesicles with itstriexponential  󿬁 tting and the statistical parameters used to check thegoodness of   󿬁 t are shown in Fig. 5. The  󿬂 uorescence lifetimes of gramicidinandTM-gramicidinareshowninTable1.Fluorescencedecayscould be  󿬁 tted well with a triexponential function. The intensity-averaged mean  󿬂 uorescence lifetimes (corresponding to emission at340 nm) were calculated using Eq. (4) and are shown in Fig. 6a. We chose to use the intensity-averaged mean  󿬂 uorescence lifetime as animportant parameter, since it is independent of the method of analysisand the number of exponentials used to  󿬁 t the time-resolved  󿬂 uo-rescence decay. Fig. 6a shows that the mean  󿬂 uorescence lifetime of the tryptophan residues in gramicidin (~3.2 ns) is lower than thatof 1-methyltryptophans in TM-gramicidin (~6.7 ns). The higherlifetime of TM-gramicidin could be due to the higher lifetime of 1-methyltryptophan compared to tryptophan [55,56]. The localizationof the  󿬂 uorophores in relatively nonpolar regions of the membranein the nonchannel conformation of TM-gramicidin could alsocontribute to the higher lifetime since lifetimes tend to be shorterin polar environments due to faster deactivation processes [66].The change in themean 󿬂 uorescence lifetime of gramicidin andTM-gramicidin as a function of increasing emission wavelength is shown inFig. 6b. The mean  󿬂 uorescence lifetime exhibits a considerable increasein both cases with increasing emission wavelength from 330 to EMISSION WAVELENGTH (nm) 400380360340320300 100806040200    F   L   U   O   R   E   S   C   E   N   C   E   I   N   T   E   N   S   I   T   Y   (   A   R   B   I   T   R   A   R   Y   U   N   I   T   S   ) 400300200100    F   L   U   O   R   E   S   C   E   N   C   E   I   N   T   E   N   S   I   T   Y   (   A   U   ) Fig. 3.  Representative  󿬂 uorescence emission spectra of gramicidin (maroon,  — ), and TM-gramicidin (blue, - - -) in POPC vesicles. The excitation wavelength was 280 nm in bothcases. Spectra are intensity-normalized at the respective emission maximum. The insetshows relative  󿬂 uorescence intensities of gramicidin and TM-gramicidin at theirrespective emission maximum. All other conditions are as in Fig. 2. See Materials and methods for other details. 300290280344340336332 63    R   E   E   S   (  n  m   )    E   M   I   S   S   I   O   N   M   A   X   I   M   U   M    (  n  m   ) EXCITATION WAVELENGTH (nm) Fig.4. Effectofchangingexcitationwavelengthonthewavelengthofmaximumemissionfor gramicidin (maroon,  ● ), and TM-gramicidin (blue,  ▲ ) in POPC vesicles. The lines joiningdatapointsareprovidedmerelyasviewingguides.Theinsetshowsthemagnitudeof REES, which corresponds to the shift in emission maximum when the excitationwavelength was changed from 280 to 307 nm (color coding is the same). All otherconditions are as in Fig. 2. See Materials and methods for other details.    M   E   A   N   R   E   S   I   D   U   E   E   L   L   I   P   T   I   C   I   T   Y   (  x   1   0   -   3    )   (   d  e  g  c  m    2    d  m  o   l   -   1    ) WAVELENGTH (nm) 260240220200 20100-10-20 Fig. 2.  Far-UV CD spectra of gramicidin (maroon,  — ), and TM-gramicidin (blue, - - -) inPOPC vesicles. The ratio of peptide to POPC was 1:50 (mol/mol) and the concentrationof POPC was 0.85mM. See Materials and methods for other details.423  A. Chaudhuri et al. / Biochimica et Biophysica Acta 1838 (2014) 419 – 428
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