73rd ISMS Champaign-Urbana: Ions Investigating Electronic and Structural Changes Imposed by Zwitterionic Pairing in Model Peptide Systems Using IR-UV Double Resonance Spectroscopy Christopher P. Harrilal, Anthony M. Pitts-McCoy, Tim S. Zwier, Scott A. McLuckey 73rd ISMS Champaign-Urbana: Ions

Introduction Zwitterionic form preferred in the condensed phase Zwitterionic interactions: strong electrostatic interaction between oppositely charge groups Salt bridges and zwitterionic pairings can largely influences secondary strucutre Existence of zwitterionic / salt-bridged systems previously questioned in gas phase Zwitterionic Zwitterionic Pairing _ + Salt-bridged interaction _ + It is well known that most amino acids exist in their zwitterionic form while in the condensed phase, and when incorporated into larger poly peptide systems strong, intramolecular, electrostatic interactions between oppositely charged groups along the peptide backbone can occur. These interactions, referred to as zwitterionic pairings and salt bridges, and can have a large influence on the secondary strucutre and function of many peptides and proteins. In the gas phase however, it is well known that all bare amino acids exist in their canonical form, which had sparked the debate of whether or not zwitterionic interactions, which are important structural elements, can exist in a gaseous environment. And for some time direct observations for the existence of these interactions had remained elusive, partly, because many of the tools commonly used to study the gas phase structures of ions only result in indirect evidence for the existence of these types of interactions. Canonical

Previous Studies IR Irradiation Multiple reported IR spectra which support zwitterionic structures for selected neutral and peptide ion systems (Dunbar, Oomens, Williams, Poutsma etc.) (Williams et al. JACS. 2009) UV Irradiation Irradiation with 157 nm promotes the loss of CO2 (Zubarev et al. Chem. Eur. J. 2006) Irradiation with 266 nm promotes photoinduced electron transfer dissociation (Julian et al. JACS. 2017 ) Spectroscopic studies using IR light performed on jet cooled neutrals and small charged peptides ions however, have shown direct evidence that these types of interactions can exist in the gas phase. And it is often seen that chelation to metal ions or water molecules can help stabilize the zwitterionic form over the canonical one. Furthermore, it has also been shown that when these interactions are present in polypeptides, they can interact with UV light to produce unique neutral losses as well form unique fragment ions, and this has been demonstrated by the Zubarev group and Julian group respectively. Taking this into account, we asked the question, if we took a polypeptide system which contained zwitterionic interactions and cooled in down to 10 K, would we see unique UV spectral signatures indicative of the presence of those zwitterionic interactions? Furthermore, can we look at the major structural differences between the conformations which exist in a zwitterionic configurations as opposed to the canonical form, provided a mixture of structures? Are there unique UV spectral signatures for zwitterionic systems that can be observed after cooling to 10 K? Major structural differences between zwitterionic vs. canonical conformations

Apparatus/Methods Spectroscopy axis MS axis Turning Quad 2 Det 1 Det 2 qtrans2 qtrans1 Cold Trap Turning Quad 1 Turning Quad 2 So we are able to do this type of experiment using a custom built, tandam, triple quad mass spectrometer that has been modified for spectroscopy. Briefly, ions are generated through nESI and guided into our second quadrupole. Here we are able to mass select the precursor ion of interest. These mass selected ions are then transferred to our cryogenically help octupole ion trap where there are collisional cooled to their vibrational zero-point levels. Once cooled we are able to overlap a UV laser with the ion packet, and if resonant photofragmentation will occur. By monitoring the fragment ion signal as a function of the UV wavelength we are able to generate a cold electronic spectrum. Once we generate this we are able to fix our UV laser on an electronic transition of interest which provides a constant fragment ion signal. At this point can then irradiated the ion packet with an IR photon, and if resonant with a vibrational transition present in the conformer which we are probing, it will effectively remove a fraction of the ground state population, thus resulting in an overall dip in the fragment ion signal. By monitoring these depletions as a function of the IR wavelength, we are able to generate a conformer specific IR spectrum. This is one of the most common ways in which we generate our data, however late in the talk I will describe an alternative method.

Generating a model system Possible zwitterionic configuration Different charge site isomers will be observed as the same m/z Unique IR Signatures Possible canonical configuration The first step in the project was to generate a model system that was capable of forming zwitterionic interactions. For this we decided to look at the plus one charge state of a custom built peptide with the sequence YGRAR. As you can see the mass of the zwitterionic configuration, where both arginines are protonated and the C-terminus is deprotonated will show up at the same m/z as the protonated canonical configuration, shown below. So using a simple mass measurement it is not possible to distinguish between these two configurations. However, as shown previously by the IRMPD community, it is possible to distinguish these charge state isomers by looking at their IR spectra. In particular, the COOH has two distinct IR signatures, the carbonyl stretch and the acid OH stretch, which are generally easily identifiably within our IR spectra. The presence of these transitions will indicate that the ions that we generate adopt a canonical configuration while the absence of both transitions strongly indicates the presence of a zwitterionic motif. So before I shown any UV data, I will first shown conformer specific IR data for the most intense UV transition of the plus one charge state of YGRAR to ensure that we are looking the ion in the correct configuration. Isomers can be distinguished in their IR spectra

IR Spectrum [YGRAR+H]+ - Hydride Stretch 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600 3700 Wavenumber (cm-1) 18 No free tyr OH Photofragment Depletion Free Asymm. NH2/ Free OH Shown here is the resulting IR spectrum in the hydride stretch region. As you can see the spectrum is quite busy, which is due to that fact that each arginine group has a total of 5 active vibrational modes. We can break the groups of transitions down into the modes that they mostly likely arise from, which is shown under the brackets, however I wont go into too much detail on that. Rather, I will focus on a few key vibrational transitions that are associated with an acid OH. The first is the highest frequency stretch, which appears at 3572 cm-1. This is generally a strong sign for the presence of an unbound acid OH. However, previous studies as well as DFT calculations show that the free asymmetric NH2 stretch of the guanidium group can show up in the same region. So there is some ambiguity as to what gives rise to this transition. On the other side of the spectrum around 3000 cm-1 there is a large broad absorption which, in the past as been associated with the acid OH group engaging in a strong hydrogen bond. However, even this part of the spectrum is ambiguous due to that fact that the signature of a free tyrosine OH which generally shows up around 3650 cm-1 is absent, which tells us that this OH group is engaged in a hydrogen bond, and may in fact be partly responsible for the broad absorption seen here. Thus, both spectral features associated with the acid OH are ambiguous, and so to tell which, if any of these transitions are caused by the presence of an acid OH we decided to do a heavy labelling experiment where we swap the 16O of the acid OH with an 18O. By doing this, any transition associated with the acid OH will experience a ~10 cm-1 shift to a lower frequency. So we are able to do this by dissolving the peptide in O-18 water and spiking in some TFA. H bonded Asym. NH2 Free Symm. NH2 Free NH H bonded OH / H bonded symm NH2 H bonded Symm. NH2 H bonded NH

18O labeled Mass Spectrum – [YGRAR+H]+ [M+H]+ 1000 2000 3000 4000 600 605 610 615 620 625 -200 -150 -100 -50 m/z Intensity Distribution of heavy labeled 18O [YGRAR+H]+ Isolation of 2 18O exchanges [YGRAR+H]+ 622 624 626 Single 18O exchange Two 18O exchange 18 The resulting mass spectrum for the heavy labelled peptide is shown on top, and as you can see both oxygens on the C-terminus are readily exchangeable. So to ensure that we will be looking at an 18O stretch we mass isolated the peptide that had undergone 2 heavy label exchanges which is shown on the bottom spectrum. 18

Comparison to 18O IR Spectrum 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600 3700 Wavenumber (cm-1) Photofragment Depletion Conformer specific IR spectrum of [YGRAR+H]+ Conformer specific IR spectrum of heavy labeled [YGRAR+H]+ The resulting IR spectrum of that mass isolated species is shown in the bottom panel. And as you can see other than a reduction in the signal to noise the spectra are nearly identical. None of the vibrational transitions which are commonly associated with the acid OH functional group experience a shift, which strongly indicates that this group is not present in this conformation. However, the absorption around 3000 cm-1 is pretty broad and some may argue that a 10 cm-1 shift would not easily be seen if the OH transition was to appear in this region. Thus, for further verification that the COOH does not exist in this conformation we also probed the carbonyl region of the IR spectrum.

Amide I/II [YAPAA+H]+ Photofragment Depletion [YGRAR+H]+ [YGRAR+2H]2+ Backbone C=O stretches Acid C=O [YGRAR+H]+ Photofragment Depletion Acid C=O clearly missing The first spectrum shown here serves as a reference spectrum. It was taken on the peptide system YAPAA, and belongs to a conformation which we have previously assigned. This conformation does have a COOH, and as you can see the acid carbonyl stretch appears as the highest frequency transition, followed by the backbone carbonyl groups. Generally, we see this acid carbonyl group appear anywhere between 1725 and 1800 cm-1 depending on its local environment. When we take a look at the spectrum for YGRAR we see that this region of the spectrum is completely empty. The highest frequency stretch appears around 1710 cm-1 right around where the backbone carbonyl stretches show up. Furthermore, this is also where the NH2 bending modes of the guanidinium begin to appear which may account for the intensity of this transition. In this last spectrum we see that by increasing the charge state to the plus 2, that carbonyl stretch appears again. Thus, from this region of the spectrum we can confirm that there is no evidence for an acid carbonyl group, and compounded with the lack of evidence for the acid OH stretch gives us strong evidence that this particular conformation does not contain an COOH group, indicating that it adopts a zwitterionic configuration. [YGRAR+2H]2+ Acid C=O 1400 1450 1500 1550 1600 1650 1700 1750 1800 Wavenumber (cm-1)

Lowest Energy Structures Fully converged conformational searches performed using the OPLS3 and Amberstar forcefields DFT optimized structures with bound tyrosine OH (~200) Large energy difference between global minimum and next highest energy conformation B3LYP 6-31+G* GD3BJ 10 8 6 Relative Free Energy ΔG298K (kJ/mol) + We have began conformational searches such that we can fit our experimental data to a strucutre. Based on the experimental observations we ran the search with the C terminus deprotonated and the two arginines protonated. Currently, we have performed two conformational searches using the OPLS3 and Amberstar force fields. From the fully converged searches, we have picked out the structures which have the tyrosine OH in a bound position, as indicated by the experimental data. From there we further optimized those structures at a high level of DFT. Shown on the left is an energy level diagram extending up 10 kJ/mol higher than the global minimum. As indicated by the diagram there is a large energy discrepancy between the global minimum and the next highest energy strucutre, which may be do to the fact that we have excluded some structures from being further optimized. Looking at the global minimum, we see that this conformation clearly adopts a salt bridge configuration where the COO- is solvated by the two charged guanidinium groups as well as that tyrosine OH group. 4 _ 2 +

Global Minimum Fit Scaled by .96 Wavenumber (cm-1) 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600 3700 Wavenumber (cm-1) AS1 AS2/AS3 AS4 Sm1 Free NH AS5 C5 Sm5 C7 C9 Sm2 Tyr. OH Sm3 C14 Sm4 1 2 3 4 Free NH 5 Free NH C5 Photofragment Depletion Shown here is the scaled calculated vibrational intensity of the global minimum strucutre compared to the experimental spectrum. Since each NH2 group has a symmetric and anti-symmetric stretch associated with it, I’ve labelled each NH2 group 1-5. Their respective symmetric and anti-symmetric stretches are indicated on the stick spectrum as Sm and AS, respectively. Calculated Intensity Scaled by .96

Global Minimum Fit Wavenumber (cm-1) Scaled by .96 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600 3700 Wavenumber (cm-1) Photofragment Depletion Calculated Intensity 1 2 3 4 Free NH 5 Free NH C5 In order to gauge how well the calculated vibrational spectrum for this strucutre matches the experimental data I’ve added drop lines to help guide the eye. We see that we have an overall good agreement between the theory and experiment for our current global minimum strucutre. However, seeing as we are still optimizing structures this is by no mean our final assignment. But we see thus far our salt bridge strucutre fits reasonably well. Scaled by .96

UVPD v CID [YGRAR+H]+ UVPD performed at 280.9 nm Similar CO2 loss as shown by the Zubarev group using 157 nm Similar formation of c ions as shown by Julian group using 266 nm Aside from probing the strucutre with IR, we also have the ability to uniquely excite this particular conformation using UV light as there is a sharp electronic transition present for this strucutre. By doing this we are able to look at the UV dissociation products resulting specifically from this strucutre. In order to generate this data we slowed the duty cycle of the experiment down, such that we could obtain unit resolution in the mass spectrum. Experimentally, we see the UV transition which belongs to this conformation appear at 280.9 nm. The top spectrum is UVPD preformed at this UV wavelength, and the bottom is the ion trap CID mass spectrum, taken on the same instrument. In the UVPD spectrum we see a neutral loss of 44, which is similar to that shown by the Zubarev group. This loss corresponds to the loss of CO2 and is clearly absent in the ion trap CID data. Lower in mass we also see a minor formation of the c4 ion which is consistent with that of the Julian work, and again we see that this is clearly absent in the CID data as well. These unique fragmentation pathways have both been shown to be present in peptides with zwitterionic interactions which, when taken into account with the IR data further supports the notion that the conformer which we are probing adopts a zwitterionic configuration. So with all this supporting evidence I can finally show some of the UV data.

UV Spectrum – [YGRAR+H]+ 35200 35300 35400 35500 35600 35700 35800 35900 36000 Photofragment Yield Wavenumber (cm-1) Laser blocked A 0.160 mJ UV power (M+H)+ (M+H)+* S0 S1 Fix IR Scan UV Photofragment Enhancement Δ𝑡= 𝑡 𝐼𝑅 − 𝑡 𝑈𝑉 = 100 ns So this is the first UV spectrum that we took of the YGRAR system, which is under the low fluence conditions that we normally take all our linear action spectra. As you can see this is a pretty rough spectrum which shows this broad absorption which is due to photofragmentation and is present as soon as the laser is unblocked. In fact the broad absorption follows the power curve and the electronic transitions resulting from the excitation of the tyrosine chromophore shows up as a relatively weak transition, nearly at the same signal level. Overall, the total photofragment yield is quite low and the UV spectrum is rather uninformative. And under these conditions there is not much that we can do as far as taking IR spectra. So in order to increase the photofragment yield we used a technique where we irradiate the ion packet with multiple IR photons right after the initial UV absorption event. In doing so we are able to increase the fragmentation yield of the tyrosine sidechain loss. This technique has been demonstrated before using a CO2 laser however works in a similar manner with an OPO provided that the IR photon is resonant with an IR transition in the excited state. Using this technique we are able to fix the IR wavelength and scan the UV to generate a IR enhanced UV spectrum.

UV + IR Enhancement [YGRAR+H]+ 35200 35300 35400 35500 35600 35700 35800 35900 36000 Photofragment Enhancement Wavenumber (cm-1) A 0.160 mJ UV power Triple Resonance Scheme (M+H)+ (M+H)+* S0 S1 (3) Fix IR(2) (2) Fix UV (1) Scan IR(1) Multiple conformers present Vibrionic transition Looking at the IR enhanced UV spectrum we can see that we obtain a much better signal to noise ratio, in fact we get a 25 fold increase in fragmentation yield when resonant with the electronic transition labelled (A). Of course you can also see that the broad absorption also gets enhance albeit by significantly less. It is also worth noting that this UV spectrum was taken under the same UV power conditions as shown on the previous slide. Looking at this UV spectrum we can see that we in fact do have multiple conformations present. So far we have only probed the transition labelled A and the second transition which we have attributed to as a vibrionic band of A. With this enhancement scheme we are able to irradiate the ion packet with a separate IR photon prior to the UV absorption, as previously described and dip the IR enhanced signal. In this scheme we are scanning the first IR photon, and fixing the UV and IR photons on transitions of interest. Using this triple resonance scheme is in fact how we took most of the IR data shown previously. Laser blocked Δ𝑡= 𝑡 𝑈𝑉 − 𝑡 𝐼𝑅(1) = 100 ns Δ𝑡= 𝑡 𝐼𝑅(2) − 𝑡 𝑈𝑉 = 100 ns

UV – [YGRAR+H]+ 1.40 mJ UV power A 35200 35300 35400 35500 35600 35700 35800 35900 36000 Wavenumber (cm-1) A 1.40 mJ UV power Increasing the UV power also enhances electronic transitions Peak widths indicate that transitions are not saturated Broad absorption also increases Is the zwitterionic motif responsible for the background? Photofragment Yield After performing those triple resonance experiments, we decided to simply increase the power of the laser and we see that the photofragment yield from the sharp electronic transitions are increased significantly such that the progressions can clearly be observed, however we also see that the large background absorption also increases significantly. We can also see just by looking at the relative peak widths that we are not saturating any of the electronic transitions significantly. At this point, after see this large absorption we thought that this may be cause by the presence of the zwitterion ionic interactions present in this system. So to test this we decided to replace the tyrosine with an alanine, thus getting rid of the aromatic chromophore. In doing this, if the large background signal still persists it would suggest that this signal is coming from the zwitterionic interactions absorbing light. Laser blocked

UV - [AGRAR+H]+ 1.40 mJ UV power 35200 35300 35400 35500 35600 35700 35800 35900 Photofragment Yield Wavenumber (cm-1) 1.40 mJ UV power Removing the tyrosine still results in UV absorption All sharp electronic absorptions due to tyrosine are absent After taking the UV spectrum of the AGRAR system in the plus one charge state we see that we get the similar broad absorption as in the YGRAR system however, all sharp electronic transitions due to the tyrosine chromophore are missing, as we would expect. Seeing this strongly indicates that the zwitterionic interactions do absorb light directly, however, just to be sure we decided to methyl esterify this system which would effectively remove all chances for any zwitterionic interaction to be present. Laser blocked

UV - [AGRAR-OMe+H]+ 1.40 mJ UV power 35300 35400 35500 35600 35700 35800 35900 Wavenumber (cm-1) Laser blocked Photofragment Yield 1.40 mJ UV power Similar broad absorption still present even after methyl esterifying C-terminus Indicates that the broad absorption cannot entirely be attributed to zwitterionic interactions Looking at the methyl ester version of AGRAR we see that the large background absorption still persists. Which at this point tells us that the background cannot entirely be caused by the presence of the zwitterionic interactions, and that some other chromophore must exist which is the main contributor. So at this point we took a step back and methyl esterified the original YGRAR system

UV – [YGRAR-Ome+H]+ 1.40 mJ UV power Broad absorption still present however, to a lesser degree Protonated guanidium group may act as a chromophore Photofragment Yield Looking at the methyl ester version of the YGRAR system we see that the background absorption decreases significantly however, it still persists even in this system. So at this point we began to think that maybe the protonated arginine may be able to act as a weakly absorbing chromophore Laser blocked 35300 35400 35500 35600 35700 35800 35900 36000 Wavenumber (cm-1)

UV+IR Enhancement [YGRAR+2H]2+ 35400 35500 35600 35700 35800 35900 36000 36100 Photofragment Enhancement Wavenumber (cm-1) A D Most dramatic reduction in the broad absorption signal B C So we looked at the plus two charge state of the YGRAR and in fact saw the largest reduction in the background absorption signal. So at this point we are still not sure what exactly is giving rise to this absorption signal, however it is apparent that it is not entirely due to the presence of zwitterionic interactions, and that it seems to be related to the number of proton and the number of arginine residues.

Conclusion [YGRAR+H]+ can serve as a model zwitterionic system Broad UV absorption is observed in [YGRAR+H]+ Broad UV absorption is reduced by methyl esterifying C-terminus but not eliminated Largest reduction broad UV absorption signal seen in the [YGRAR+2H]+2 Currently no clear UV spectral signature for zwitterionic motifs Broad UV absorption seems to be related to number of protons vs. number of arginine residues

Acknowledgements 2018 Zwier Research Group National Science Foundation (NSF CHE 1465028)

Amide I/II Global Minimum Fit Photofragment Depletion Tyr C=O NH2 bend / C=O (Arg1) st. Arg2 /Gly1 NH bend Gly C=O Ala C=O CO2- bend Arg1 NH bend Calculated Intensity Arg NH2 CO2- AS Arg NH2 Ala NH bend Arg NH2 1400 1450 1500 1550 1600 1650 1700 1750 1800 Wavenumber (cm-1) Scaled by .97

IR Spectra Amide I/II – [YGRAR+2H]2+ Transition A Transition B Photofragment Depletion Transition C 1400 1450 1500 1550 1600 1650 1700 1750 1800 Wavenumber (cm-1)

IR-UV Hole Burn on COOH C=O Stretch D C Photofragment Signal 35400 35450 35500 35550 35600 35650 35700 35750 Wavenumber (cm-1)

UVPD AGRAR v AGRAR-OME AGRAR Intensity Intensity AGRAR-OME m/z -42 -60 1.0 AGRAR 0.8 -18 Intensity 0.6 -42 0.4 y4-NH3 -60 -H2O y4 a3 0.2 b3+1 y4 – 42 z2 0.0 0.0 b3+1 -0.2 y1 x3 y1-NH3 y2-NH3 -0.4 z2 y2 Intensity a3 -0.6 -42 -0.8 AGRAR-OME y4-NH3 y4 -1.0 100 150 200 250 300 350 400 450 500 m/z

UVPD of 18O heavy labelled YGRAR 1 2 3 4 5 400 450 500 550 600 650 -8 -7 -6 -5 -4 -3 -2 -1 Intensity m/z No exchanges [M+H]+ -107 2 exchanges -107 Shows that the tyrosine OH does not readily exchange [M+H]+ (2 18O exchanges)

Possible Charge Site Isomer Responsible for Broad UV Absorption

Possible [YGRAR+2H]2+ Charge Site Isomer

[Ac-YGRAR+H]+ 1.40 mJ Wavenumber (cm-1) 35300 35400 35500 35600 35700 35800 35900 Wavenumber (cm-1)

YGGFLK Photofragment Yield Wavenumber (cm-1) 35500 35600 35700 35800 x4 ion coming from excitation of the shared proton? Spectroscopy on Ac-YGARA to see the influence of coo- functionality without shared proton being present UVPD of YGGFLK Spectroscopy on Ac-YGGFLK

Only 4 NH Stretches, One Nearly Degenerate? Free Acid 2800 3000 3200 3400 3600 Wavenumber (cm-1) Photofragment Yield Broad NH3 Only 4 NH Stretches, One Nearly Degenerate? Free Acid Free Tyr OH

1400 1500 1600 1700 1800 1900 Intensity Wavenumber (cm-1)