over the past decades 
 our atomistic understanding of protein structure 
 and function has been revolutionized by x - ray crystallography and 
 nmr spectroscopy , and magnetic resonance techniques have provided 
 information on the time scales of protein conformational dynamics . 
 however , until recently , no direct experimental techniques existed 
 that could combine atomistic structural information with time resolution 
 sufficient to observe the intrinsic functional time scales of many 
 proteins . among the new approaches to studying protein conformational 
 dynamics , 
 infrared ( ir ) spectroscopy of amide i vibrations ( the amide carbonyl 
 stretch between 1600 and 1700 cm ) has seen a surge 
 of interest as a result of advances in 2d ir spectroscopy and structure - based 
 modeling , which provides an atomistic interpretation of protein ir 
 spectra . amide i vibrations are delocalized , involving the concerted vibration 
 of numerous coupled carbonyls of peptide units of the protein backbone . the sensitivity of the coupling between carbonyl groups to their relative 
 position and orientation in space means that the amide i band is influenced 
 by the underlying symmetry , size , and backbone structure of the protein . though interpretation of protein ir spectra is often complicated by 
 the presence of many broad peaks in a small frequency window , a new 
 generation of atomistic spectral modeling that draws from molecular 
 dynamics ( md ) simulations of the protein provides new avenues by which 
 to interpret these experiments . in this context , the molecular origin of the observed high ion 
 throughput ( > 10 ions s ) and highly 
 selective transport of k ions of na ( > 1000:1 ) in the bacterial potassium channel kcsa is still 
 hotly debated . traditional models of the selectivity 
 and transport assume the selectivity filter is inflexible and that 
 differences in the ion binding can be explained by a geometry optimized 
 to bind k ions ( the  snug - fit  model ) . this is in contrast to other proposed explanations , where significant 
 fluctuations in the selectivity filter are invoked . more recently , simulations have suggested the assumption 
 of alternating k ions and water molecules transporting 
 through the selectivity filter may be incorrect , and the k ions are more densely packed than previously thought . what is clear , however , is that understanding 
 the behavior of the selectivity filter is essential to understanding 
 k transport in kcsa , which requires experimental characterization 
 of the dynamical interaction between the ions and the selectivity 
 filter . ( a ) structure of the transmembrane region of kcsa highlighting 
 the extended filter region ( red ) and k ions in the ( s1 , 
 s3 ) configuration ( purple ) . ( b ) schematic illustration of the ion 
 occupancy within the selectivity filter for the three configurations 
 investigated . the tvgyg structural 
 motif constituting the selectivity filter 
 is a highly conserved sequence among potassium channels . each subunit of the kcsa tetramer consists of 
 an intracellular gating domain and a 15  narrow pore 
 domain in which the selectivity filter resides ( figure 1a ) . the carbonyl groups in this 20-residue filter define a 
 set of binding sites , labeled s1s4 , in which k binds in an 8-fold coordinated geometry ( figure 1b ) . x - ray crystallography indicates that under physiological 
 concentration k can bind in one of two configurations , 
 either at sites s1 and s3 or at sites s2 and s4 . water is assumed to occupy the respective other binding 
 sites for each configuration , though concerns have been raised over 
 whether this is the case during transport . na has been observed to bind primarily in an in - plane 
 position ( figure 1b ) , though there is still 
 some debate over which residues coordinate na . here we demonstrate on three well - characterized 
 examples of ion - binding 
 in kcsa that the ion - induced conformational changes of kcsa can be 
 characterized using amide i ir spectroscopy in combination with computational 
 modeling . using attenuated total reflection ( atr ) ir spectroscopy , 
 it was recently shown that significant differences in the amide i 
 spectrum can be observed when the buffer cation composition is varied 
 between k and na , but the origin of these 
 differences remained elusive . here we 
 present new difference ir spectra between k and na buffers designed to prepare the binding configurations pictured 
 in figure 1b and use structure - based spectroscopic 
 modeling that draws on md simulations to interpret the data . our analysis 
 reveals the sensitivity of amide i vibrations of the selectivity filter 
 and pore region to the conformational changes which occur on binding 
 k or na . the full - length kcsa 
 channel ( 167 amino acid length monomer ) , containing a 6xhis - tag inserted 
 directly after met1 and cloned into prsf1 ( merck ) , was expressed in escherichia coli bl21 ( de3 ) cells on induction with 1 mm 
 iptg ( isopropyl--d - thiogalactopyranoside ) . bacteria 
 were collected and resuspended in buffer a ( 100 mm nacl , 50 mm kcl , 
 50 mm sodium phosphate , ph 8.0 ) solution and lysed by sonication in 
 the presence of a protease inhibitor cocktail ( complete edta - free , 
 roche ) . the protein was solubilized by 1 h incubation with 20 mm n - dodecyl--d - maltopyranoside ( ddm , avanti ) 
 which is known to not interfere with the protein s spectral 
 features in the amide i band , followed by centrifugation at 30000 g for 30 min . the supernatant was incubated with co affinity resin ( thermo ) in the presence of 20 mm imidazole 
 for 1 h at 4 c , loaded onto a column , and washed with 40 mm 
 imidazole and 1 mm ddm in buffer a. the protein was eluted with 400 
 mm imidazole and 1 mm ddm in buffer a and extensively dialyzed versus 
 nacl buffer b ( 150 mm nacl , 1 mm ddm , 10 mm hepes , ph 7.5 ) to remove 
 k ions . to assist with sample transparency in the 
 infrared , all exchangeable protons of kcsa were deuterated by multiple 
 concentration  dilution cycles with d2o - based buffer 
 b in a 10 kda mwco spin concentrator ( amicon , millipore ) over the 
 course of several days until the h2o content was below 
 1% as determined by absorption at 3400 cm on a 
 ftir spectrometer ( thermo ) . temporary heating of the sample to 37 
 c and subsequent incubation at 4 c ensured proper deuteration 
 of the buried parts of the protein . the desalted and deuterated kcsa 
 was concentrated to 78 mg / ml , frozen in liquid n2 , and stored at 80 c until further use . correct secondary 
 structure of kcsa in k - free buffer b was confirmed via 
 cd spectroscopy . the fraction of correctly folded tetrameric protein 
 was estimated by loading unexchanged samples of kcsa on a normal sds - gel , 
 and from the densiometry of the monomeric and tetrameric band at approximately 
 18 and 75 kda , it was estimated that > 95% of the sample was composed 
 of correctly folded tetrameric protein . to introduce k ions into the sample 
 in a controlled fashion , the kcsa was 
 subjected to further concentration  dilution cycles with a modified 
 buffer b ( 150 mm kcl , 1 mm ddm , 10 mm hepes pd 7.9 in d2o ) . for the ftir absorbance spectra , 
 approximately 2.5 l of kcsa sample in different salt conditions 
 was sandwiched between two 1 mm thick caf2 windows ( cening 
 optics ) , separated by a 50 m ptfe spacer . for each sample , 
 2048 averages were collected on a nicolet 380 ftir spectrometer ( thermo 
 scientific ) at 1 cm resolution against a background 
 of dry air at 20 c . ftir spectra of each buffer , without protein , 
 were also collected in order to subtract the background d2o absorption from the kcsa spectra . difference spectra are calculated 
 as ftir spectra of ( kcsa in kcl , buffer subtracted ) ( kcsa 
 in nacl , buffer subtracted ) . in 
 order to obtain a molecular interpretation of the spectral features 
 above , we used a model of the amide i vibration that maps structure 
 vibrational frequencies and couplings between oscillators . this procedure draws on md simulations of kcsa for each ion - binding 
 configuration of interest and uses it to parametrize a quantum - mechanical 
 local mode hamiltonian for the coupled peptide units of the protein 
 backbone , which govern the vibrational spectroscopy of the amide i 
 band . md simulations were 
 performed using the gromacs software package and the charmm36 force 
 field . we started with the full - length tetrameric kcsa crystal structure 
 by uysal et al . ( pdb accession code 3eff ) and embedded this 
 in a dmpc membrane using the g_membed function in 
 gromacs . the protein this system was equilibrated 
 for a total of 2 ns ( 1 ns nvt equilibration , 1 ns npt equilibration ) , 
 before running a further 7 ns trajectory to use as a basis for the 
 spectral calculations . all stages of the simulation were run with 2 fs time steps , nos  hoover 
 thermostat , semi - isotropic parrinello rahman pressure coupling 
 ( where appropriate ) , and the lincs constraint algorithm . to compare 
 the effects of different salts on the spectra , we replaced the contents 
 of the selectivity filter prior to equilibration with either 2 k ions and 2 water molecules ( with k in either 
 s1 , s3 or s2 , s4 , water occupying with remaining sites ) or 2 na and 2 water molecules ( using the positions from thompson 
 et al . ) . during the md production run , the k ( na ) ions were held in place with harmonic 
 position restraints . individual peptide groups and their associated amide i 
 transition dipoles are identified in structures sampled from md simulations . a local mode hamiltonian is parametrized from the structure using 
 the molecular electric field to set the diagonal site frequencies 
 and off - diagonal couplings between sites . the delocalized eigenstates 
 and their corresponding transition dipole moments are used to calculate 
 the ir spectrum and use doorway modes to visualize the vibrations . figure 2 illustrates the simulation protocol 
 used to link the experimentally 
 observed ir spectra with md simulations , as reviewed in detail in 
 ref ( 10 ) . the local 
 mode hamiltonian is constructed in the basis set of the 668 backbone 
 peptide units and was parametrized using frozen configurations of 
 the protein and water . the structure determines the position and orientation 
 of each peptide unit , and the corresponding amide i transition dipole 
 moment for that site is positioned in the plane of the peptide unit 
 between the carbon and oxygen . the diagonal elements of the hamiltonian 
 are the vibrational transition energies ( site frequencies ) between 
 the  = 0 and  = 1 states of the amide i vibration . hydrogen - bonding 
 interactions with the carbonyl oxygen downshift these frequencies 
 by an amount that depends on the local electrostatic environment . 
 for this calculation , we used the empirical spectral map in which 
 the frequency of each oscillator is correlated with the electric field 
 acting on the oxygen of the peptide unit along the carbonyl bond developed 
 by reppert et al . the hamiltonian s 
 off - diagonal elements , or couplings , are determined by the spatial 
 configuration of the peptide units . in the case of through - space interaction , 
 we used transition dipole coupling , which depends on the distance 
 and angle between the two oscillators . for bonded peptide units , the through - bond coupling was obtained 
 from the nearest - neighbor coupling model developed by jansen et al . , which maps the  and  angles of 
 the bonded peptide units onto a coupling . diagonalizing this hamiltonian 
 led to eigenstates and eigenvalues which were used to calculate an 
 ftir spectrum of individual configurations of protein and water . since 
 parameters used depend intimately on the sub - angstrom configuration 
 of water and protein , we generate hamiltonians for structural snapshots 
 at 1 ps intervals of the trajectory . in the case of the k calculations , an average of the s1 , s3 and this approach is known 
 to qualitatively reproduce spectral features . while it does not quantitatively 
 reproduce exact peak frequencies , line widths , or absolute intensities , 
 the relative frequency shifts and intensities of resonances are reliable 
 metrics for comparison . our calculations also neglect side chain vibrations 
 that may absorb in the 16001700 cm region , 
 such as the amide vibrations from glutamine and asparagine and the 
 cn vibrations of arginine . while side chains certainly contribute , 
 it is at a significantly lower level than amide i , and their frequencies 
 are well - known for the interpretation of experimental spectra . although 
 the spectral map we use here neglects side chains , it is important 
 to note that other maps have been developed which are developed with 
 the aim of capturing the vibrations of side chain amide groups ( asp , 
 glu ) , though the application of this 
 maps to side chains in larger proteins has not been characterized . the diagonalization of the local 
 mode hamiltonian provides not only the energies of the vibrational 
 modes but also the corresponding eigenstates . interpretation of these 
 eigenstates , however , is hampered by the sheer number of them ( > 30  000 
 in this case ) , even for a small subset of the protein structure at 
 a reduced sampling rate . analogously to how principal component analysis 
 can be used to describe which input variables are principally responsible 
 for observed trends , doorway mode analysis shows which amide units 
 contribute most significantly to the ir spectrum in a specific frequency 
 window . to obtain a set of representative 
 strongly ir - active states from a subset of eigenvectors , we apply 
 the doorway mode analysis in ref ( 34 ) , using a  3 cm window 
 about the stated center frequency . though outlined in detail 
 in ref ( 34 ) , we briefly 
 review doorway mode analysis . the aim is to find a transformation 
 matrix i , which will transform 
 vectors in the site basis to the doorway basis . the doorway mode basis 
 is one where most of the intensity of the transition is contained 
 within the first three modes . first , we define the matrix ti , a subset of the transformation 
 matrix obtained from the eigenvalue decomposition of the hamiltonian . the subset is defined to only include eigenstates with eigenvalues 
 within the frequency range ri . a singular value decomposition ( svd ) of the eigenstates of 
 the hamiltonian transforms them to a basis where each subsequent component 
 contributes less to the transition intensity . combining the svd transformation 
 matrix ui with our eigenstate 
 transformation matrix ti yields the overall transformation matrix i = uiti . with this matrix , as shown in figure 3 , the ftir spectra of 
 kcsa in both kcl and nacl 
 show a broad peak centered at 1655 cm , consistent 
 with the high -helix content of the protein . the asymmetric 
 line shape features a broad shoulder at lower frequencies , which includes 
 contributions from the side chain absorptions of asp , arg , gln , tyr , 
 trp , and gln residues . to specifically 
 capture the spectral changes which arise solely from the binding of 
 ions by the selectivity filter , we focus on the analysis of the difference 
 spectra , i.e. , the spectrum of kcsa in kcl buffer minus the spectrum 
 of kcsa in nacl buffer . the ftir difference spectrum , in figure 3 , is highly structured , with several distinct features . 
 of these features , we select three for analysis ( labeled 1 , 2 , and 
 3 ) because they are the largest amplitude differences within the amide 
 i window , are not overlapping other features , and are well - reproduced 
 by our calculations . peak 1 is a strong and broad negative peak at 
 1625 cm , peak 2 is a strong positive band at 1660 
 cm , and peak 3 is a less intense narrow positive 
 band at 1680 cm . given that the amide i vibrations 
 of 668 peptide units contribute to the full ftir spectra , and that 
 only 16 carbonyls are in direct contact with the bound ions at any 
 one time , it is not surprising that the largest spectral changes between 
 kcl and nacl buffers are on the order of 4% . nevertheless , this change 
 is large enough to suggest that a significant number of amide carbonyls 
 are sensitive to the identity of the bound ion . experimental spectra 
 of kcsa with kcl or nacl buffers and the difference 
 spectrum ( kcl  nacl ) illustrating the correspondence of key 
 features . our experimental ftir spectra 
 and difference spectrum are consistent 
 with the difference atr infrared spectra reported recently by futurani 
 et al . our peak positions differ by < 5 
 cm , but this readily explained by our use of deuterated 
 sample as opposed to experiments in h2o , and the differing 
 chemical environment of the sample ( ddm micelles in this work versus 
 asolectin bilayer ) . simulated 
 ftir and ftir difference spectra , shown in figures 4a and 4b , respectively , are able to 
 qualitatively reproduce the key experimental features . the full ftir 
 center frequency and line width are in good qualitative agreement , 
 though as noted earlier , quantitative agreement should not be expected 
 from these calculations . the experimental line shape is not fully 
 reproduced by calculation , notably in the 16201650 cm region , in which there is a shoulder in the experimental 
 spectra which is not captured in the calculations . we attribute this 
 primarily to side chain absorptions in this region ( namely arg at 
 1605 cm , tyr at 1615 cm , trp 
 at 1618 cm , and gln at 1640 cm ) , which are not included in our calculation 
 since vibrational frequency and coupling models do not presently exist . comparison 
 of the experimental and calculated spectra for kcsa 
 in kcl buffer ( a ) and the difference spectra ( b ) . peaks 1 , 2 , and 
 3 ( see main text ) are highlighted in red , green , and blue respectively , 
 and the side chain region is highlighted in orange . in the calculated ftir difference spectrum , we 
 find the three features 
 highlighted in the experimental data are reproduced well both in peak 
 frequency ( differing by only a few wavenumbers ) and in relative intensity . the reproduction of both the frequency and intensity in our calculations 
 suggests the nature of the delocalized vibrational modes is well captured . the most notable region of mismatch between the experimental and 
 calculated difference spectra is again the 16201650 cm region . while this could reflect a limitation of 
 the vibrational coupling model , is it more likely to reflect the neglect 
 of side chain vibrations in our calculations . previous work has shown 
 that the y78f mutant , which shows similar functional behavior to the 
 wt protein , gives rise to a difference 
 spectra distinct from the wild - type , with the most notable differences 
 being in the 16201650 cm region . however , we note that , while the side chain 
 absorptions may indeed contribute significantly to the experimental 
 difference spectrum , our analysis focuses on peaks which are clearly 
 reproduced by the carbonyl - only modeling , indicating that a discussion 
 of these peaks in terms of delocalized amide i vibrations only is 
 appropriate . comparison of calculated difference spectra for different 
 subsections 
 of kcsa . the filter - only calculation is unable to reproduce the features 
 observed in the extended filter region , the full transmembrane region , 
 and the experimental spectra . to determine the participation of different regions of the 
 protein 
 to the spectral features observed , we calculated difference ftir spectra 
 using various subdomains of the full tetrameric protein complex . these 
 sections are ( a ) the four tvgy chains comprising the selectivity filter , 
 ( b ) the  extended filter region comprising the selectivity 
 filter and the four pore helices , and ( c ) the entire transmembrane 
 region of the protein . we found that the calculated ftir spectra for 
 the transmembrane region and the extended filter differed only in 
 the intensity of some features but were otherwise consistent with 
 each other . the selectivity filter alone could not reproduce the difference 
 spectra , notably the 1660 cm peak , suggesting 
 the larger protein complex and coupling between the monomer units 
 are actively participating in generating the observed experimental 
 spectra . calculated spectra for the extended filter region for the various 
 ion - binding configurations . the calculated difference spectrum is 
 shown in gray . in order to elucidate 
 the origins of the features in the difference 
 spectra , we show the calculated ftir spectra for the individual ion - binding 
 configuration of the extended filter region in figure 6 . from this , we see that peaks 1 and 3 come from distinct 
 peaks in the na- and k - bound calculations , 
 respectively , while peak 2 comes from a change in intensity and line 
 shape of a peak present in the kand na calculations . taken in conjunction with the calculations 
 on truncated regions of the protein , this suggest the 1660 cm peak in the difference spectra ( peak 2 ) arises from 
 changes in the -helices surrounding the pores . the nature of 
 these structural changes is discussed below . to visualize the molecular 
 origins of the vibrations which contribute 
 to the difference spectrum , we apply a doorway mode analysis to our 
 calculated eigenvectors for the extended filter region . the first at 1630 cm corresponding to peak 1 , 1660 cm ( peak 2 ) , 1674 
 cm ( peak 3 for s1 , s3 binding ) , and 1680 cm ( peak 3 for s2 , on the basis of this calculation , the four frequencies can be assigned 
 to four modes with distinct vibrational motions involving amide units 
 within the extended filter region ( see figure 7 ) . the amide units contributing most significantly to the 1630 cm mode , designated the b - site mode , 
 are t75 carbonyls with some contribution from the carbonyls of the 
 pore -helices . these carbonyls and the direction of their transition 
 dipoles are identified with outlined arrows in figure 7a . carbonyls on opposite monomers of the filter mode vibrate 
 out - of - phase , contracting on one side and extending on the other ; 
 however , the net transition dipole moment of all oscillators constructively 
 adds so that the total transition dipole moment ( green arrows ) is 
 orthogonal to the filters pore . the 1660 cm mode 
 ( labeled  helix  ) is present in all cases and is constituted 
 almost exclusively by vibrations from the pore -helices . carbonyls 
 in all monomers are in - phase and contribute to an overall dipole moment 
 that is oriented along the pore ( figure 7b ) . 
 the 1674 cm mode of the ( s1 , s3 ) configuration 
 is termed  s3  , due to the strong involvement of the 
 t75 and v76 residues in the filter which define the s3 binding site 
 ( figure 7c ) . similarly the 1680 cm mode of the ( s2 , s4 ) configuration is termed s2  
 due to the large amplitude in the v76 and g77 residues which define 
 the s2 binding site ( figure 7d ) . visualizations 
 of calculated doorway modes of the extended filter 
 region presented as side view ( left ) and top view looking down the 
 pore ( right ) . color indicates the relative sign of the co vibrational 
 motion ( contraction / extension ) , while the color intensity indicates 
 its amplitude . outlined arrows indicate the direction of the transition 
 dipole moment of individual carbonyls contributing most to the mode . the direction of the net transition dipole moment for the whole extended 
 monomer (  ) is shown with dotted green arrows . the colors in 
 circles on the right indicate the overall displacement of the selectivity 
 filter carbonyls in that strand . na and k ions 
 are blue and purple , respectively . the extent of delocalization 
 determines how specific an ir resonance will be to interactions at 
 a particular site of the protein . the extent of delocalization of 
 vibrations is determined by two competing factors : the strength of 
 couplings between oscillators ( j ) and the variation 
 in individual site frequencies (  ) . increasing j and decreasing  leads to increasing delocalization . 
 in the case of the  helix  mode ( peak 2 ) ; the near - uniform 
 electrostatic environment of the -helix minimizes  
 while also locking the oscillators into a geometry which gives rise 
 to significant coupling between oscillators , resulting in a vibrational 
 mode which is spread over the length of the pore helix . we believe 
 the delocalized nature of the  helix  mode is the key 
 to why peak 2 is observed in the difference spectrum . slight changes 
 in the structure of -helices , such as a slight bending of the 
 helix from the helical axis , have been predicted to change the intensity 
 and line widths of ir peaks . the increase in intensity 
 at 1660 cm in the k bound data suggests 
 that the -helices deviate further from an ideal -helix 
 geometry than the na bound structure . the remaining 
 three modes we identify , however , are more local 
 in character , because the electrostatic environments that arise from 
 binding k and na strongly and selectively 
 influences the frequency of the coordinating carbonyls . for the b mode , the proximity of na lowers the frequency of the four t75 carbonyls by > 25 
 cm relative to the v76 carbonyls . considering 
 the weaker 
 coupling between t75 and v76 carbonyls ( j < 8 
 cm ) , binding nain plane with the t75 residues largely decouples these vibrations from the 
 rest of the extended filter , creating a site - specific infrared transition . 
 for the s3 mode , the similar frequencies of the k - binding 
 v76 and g77 carbonyls (   10 cm ) and their strong coupling ( j > 8 cm ) lead to a vibrational transition that is largely localized on the 
 eight carbonyls defining the s3 site . similar factors seem to be responsible 
 for the s2 mode which strongly involves the t75 and v76 residues . in reality , all of the vibrational motions we observe are neither 
 localized to a single oscillator nor delocalized over the entire protein 
 but have varying degrees of delocalization . furthermore , the doorway 
 modes do not reflect how these characteristic vibrational motions 
 influence one another or act in concert as would be appropriate for 
 developing a dynamical picture of the protein  ion interaction . our results show that ir spectroscopy 
 coupled with structure - based 
 spectral modeling can be used as a sensitive tool to investigate functionally 
 relevant conformational changes in large membrane proteins and , furthermore , 
 detect subtle local changes . we have demonstrated this capability 
 in this study by resolving the different environments of the binding 
 sites in the selectivity filter of kcsa and identified unique ir signatures 
 of ion binding states . structure - based spectroscopic modeling of the 
 extended filter region allowed us to perform the doorway mode analysis 
 which established the link between our experimental data and the ion - induced 
 structural rearrangements in the protein . we found different vibrational 
 couplings for the k- and na - bound states between 
 the tvgy core of the channel and the pore helices , which suggest that 
 the helices are not simply a structural support but may indeed play 
 a central role in determining the different transport properties of 
 kcsa for these ions . it has previously been suggested that the pore 
 helices may act as a mediator between selectivity filter and activation 
 gate , with particular emphasis on the mechanism of c - type inactivation . our work suggests the role of the pore helices may extend beyond 
 this and even have a role in determining the energetics and dynamics 
 of ion binding . to what extent these vibrational couplings map onto 
 functionally relevant interactions could be further studied by mutations 
 that alter the interface between the pore helix and selectivity filter . though our work was not designed to directly 
 study kcsa under transport 
 conditions , some of our conclusions have important implications in 
 this area . we find experimental evidence for strong interactions between 
 the t75 carbonyls and na . in their recent paper , this suggests 
 perhaps that na ions are not conducted , in part , because 
 of the influence of the na on the coordinating carbonyl 
 dynamics (  rigidifying  them ) . on the technical 
 side , one possible extension of our work here this will 
 capture the dynamics of the protein in real time , shedding light on 
 the underlying molecular mechanisms of the early time events in binding 
 in transport , thus far inaccessible to standard techniques . this provides 
 the tantalizing possibility that mechanisms suggested by md , such 
 as those for c - type inactivation , could be directly 
 tested .
the effect of ion binding in the 
 selectivity filter of the potassium 
 channel kcsa is investigated by combining amide i fourier - transform 
 infrared spectroscopy with structure - based spectral modeling . 
 experimental 
 difference ir spectra between k+-bound kcsa and na+-bound kcsa are in good qualitative agreement with spectra 
 modeled from structural ensembles generated from molecular dynamics 
 simulations . 
 the molecular origins of the vibrational modes contributing 
 to differences in these spectra are determined not only from structural 
 differences in the selectivity filter but also from the pore helices 
 surrounding this region . furthermore , the coordination of k+ or na+ to carbonyls in the selectivity filter effectively 
 decouples the vibrations of those carbonyls from the rest of the protein , 
 creating local probes of the electrostatic environment . 
 the results 
 suggest that it is necessary to include the influence of the surrounding 
 helices in discussing selectivity and transport in kcsa and , on a 
 more general level , that ir spectroscopy offers a nonperturbative 
 route to studying the structure and dynamics of ion channels .