Cold Ion Spectroscopy of Biological Molecules in the Gas Phase

The three-dimensional (3D) structures of proteins and peptides in vivo largely determine their biological functions. In vitro these native structures and their heterogeneity reflect a subtle balance between noncovalent intramolecular interactions and those with the surrounding solvent molecules. Decoupling the intra- and intermolecular interactions and revealing the intrinsic structures of biomolecules in the gas phase is crucial for understanding protein-peptide (-protein, -membrane) binding processes and protein folding, and can assist in silico drug design. In addition to this, the spectroscopic studies of (bio)molecules in the gas phase provide crucial information that serves as a benchmark for validation of theoretical approaches, used for structural computations. In this thesis report we demonstrate the use of conformer-selective, Cold Ion Spectroscopy (CIS), combined with mass spectrometry and electrospray ionization technique, to reveal spectroscopic fingerprints of closed-shell (bio)molecular species in the gas phase. These fingerprints provide a stringent test for validating calculated structures of the molecules. Electronic and conformer-specific vibrational spectra of charged molecules, collisionally cooled in a linear radio frequency 22-pole ion trap have been measured using photofragment detection schemes that employs UV and/or IR laser light. A detailed set of spectroscopic and structural constraints, obtained using CIS method for an antibiotic peptide, gramicidin S (GS) allows an unambiguous determination of its 3D structure in the gas phase. The calculated gas-phase structure of GS differs substantially from its known structure in the condensed phase and, thus, provides complementary information for modeling biological activity of this antibiotic. At the next step we form hydrogen-bonded complexes of GS with a gradually increasing number of water molecules. Spectroscopy of such peptide-water clusters targets to bridge the gap between the gas phase and the in vitro structures. We have shown that both, UV and IR spectra of large GS-water clusters remain vibrationally resolved, if the species are cooled to cryogenic temperatures. The observed detailed spectroscopic fingerprints of the hydrated peptides provide a benchmark for their structural analysis. While CIS method was developed for biological species, here we demonstrate its successful application for structural determination of highly-reactive catalytic metalorganic species. The experimentally observed lack of a characteristic NH stretch vibration in the IR spectra of such a species allowed an unambiguous selection of its structures from several low-energy calculated candidates. The current approach for solving structures of biomolecules implies a comparison of a measured spectroscopic fingerprint with that, calculated for a candidate structure. Unfortunately, high accuracy structural computations become time consuming and challenging for large peptides. We revealed a specificity in electronic spectra of phosphorylated peptides, which coordinates with the docking position of a phosphogroup in peptides. This observation, if confirmed for a large variety of peptides, may allow a prediction of phosphorylation sites without a call for calculations. Regarding a relative simplicity of UV spectroscopy we see a potential of this approach as an orthogonal analytical method that assists mass spectrometry in some puzzling cases of peptide analysis. At the end of this work we tried to push the limits of CIS approach towards very large species and, for the first time, measured IR spectrum of a bare protein, cytochrome c, in the gas phase.

A Combination of Cold Ion Spectroscopy and Ion Mobility for the Study of Complex Peptides and Small Proteins

Georgios Papadopoulos

We present in this work experiments that push the limits of cold ion spectroscopy to the study of complex peptides and small proteins in the gas phase. Although the low temperature attainable in a cold ion trap greatly simplifies the electronic spectra of large molecules, conformational heterogeneity can still be a significant source of congestion, complicating spectroscopic analysis. To overcome this hurdle, we propose an in tandem combination of ion mobility spectrometry and photofragment spectroscopy. This permits for conformational separation prior to laser interrogation. Towards this end, we describe a proof-of-principle experiment where we combine high- Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS) with electronic spectroscopy. We demonstrate that using FAIMS to separate gas-phase peptide conformers before injecting them into a cold ion trap allows one to decompose a dense spectrum into contributions from different conformational families. In the inverse sense, cold ion spectroscopy can be used as a conformation-specific detector for ion mobility, allowing one to separate an unresolved mobility peak into contributions from different conformational families. The doubly protonated peptide bradykinin serves as a good test case for the marriage of these two techniques as it exhibits a considerable degree of conformational heterogeneity that results in a highly congested electronic spectrum. Our results demonstrate the feasibility and advantages of directly coupling ion mobility with spectroscopy and provide a diagnostic of conformational isomerization of this peptide after being produced in the gas phase by electrospray. In a second series of studies we show the potential but also the limitations of our spectroscopic approach for investigating small, naturally occurring proteins, by applying it to ubiquitin, a protein of 76 amino acids. We present electronic photofragment spectra of various protonation states of ubiquitin and we compare our results to literature data acquired by ion mobility spectrometry. These experiments show that the information obtained by laser interrogation of molecules of this size is limited and could be enhanced by a combination with ion mobility techniques. Although some of the results obtained may be important in characterizing small proteins in the gas phase, these experiments are at an early stage and the application of photofragment spectroscopy on proteins needs more investigation.

EPFL2012

Spectroscopic Probes of Conformational Isomerization of Biological Molecules in a Cold Ion Trap

Caroline Seaiby

The function of biologically active molecules depends both on their structure and on their conformational dynamics. In solution, intramolecular interaction with the solvent will influence different biological processes, i.e. protein folding. However secondary structure (helices, sheets) is heavily influenced by intramolecular forces and hence it is important to isolate biological molecules in order to study their intrinsic behavior. Moreover, gas phase studies on biomolecules provide critical information that can serve as benchmarks to test the accuracy of theoretical predictions. The main focus of this thesis is the investigation of the potential energy surfaces of biomolecules in the gas phase. Biomolecular ions are produced in the gas phase via nano-electrospray, mass-selected and guided into a cold, 22-pole ion trap where they are cooled via collisions with cold helium. A variety of double-resonance techniques based on photofragmentation detection are then applied to obtain information on the molecule stable conformations, the potential barriers separating stable conformations and their connectivity. We applied these techniques on molecules of increasing size, starting with protonated phenylalanine and proceeding to the 7amino acid peptides, Ac-Phe-(Ala)5-LysH+ and the 12-residue peptide Ac-Phe-(Ala)3-(Gly)4-(Ala)3-LysH +. As a first step, electronic spectra and conformation-specific IR-UV double resonance spectra are measured. These measurements, in combination with DFT calculations, allow the assignment of two stable conformers in the case of the protonated phenylalanine and four in the case of the seven residue peptide. In the second part of this work we focused on the conformational isomerization of these molecules by performing infrared and ultraviolet hole-filling spectroscopy in the ion trap. We demonstrated that we can induce isomerization between stable conformers of each molecule via vibrational excitation. After energy dissipation, the excited molecules are redistributed among the initially identified conformers, but no new minima were detected. In the last part of this thesis, the fractional populations and the isomerization quantum yields are determined through infrared induced population transfer spectroscopy. Protonated phenylalanine reveals conformational selectivity in its isomerization while the relaxation of the infrared excitation leads to the equilibrium distribution in the case of the 12-residue glycine-containing peptide. The steps that occur during the energy dissipation are discussed in this thesis.

EPFL2011

Influence of the protein environment on spectroscopy and ultrafast dynamics of retinal in bacteriorhodopsin

Selma Schenkl

The environment is important for the exact course of a chemical reaction. As an example of the strong influence of the environment on the reaction, this work studies the isomerization reaction of the chromophore retinal in the binding pocket of the protein bacteriorhodopsin. Three selected distance scales with reference to the chromophore are addressed: The distant protein surface at a length scale of 3-5 nm, the fuzzy environment of the binding pocket (1-2 nm) and the immediate neighborhood represented by the amino acid tryptophan Trp86 (5 Å). First, static absorption and fluorescence spectroscopies are applied to three dimensionally crystallized bacteriorhodopsin. An especially adapted set-up was developed for each task. The analysis of data is carried out in comparison with the measured spectra of the native protein membrane in solution. While the absorption spectrum shows a broadening on the high-energy side, the fluorescence spectrum deviates in the general shape of that measured in solution. The synoptic analysis of all spectra reveals the existence of three spectroscopically distinct species in the crystal: BR490 (absorption maximum at 490 nm) with a relative contribution of 28%, BR570 (native spectrum of solution, 68%), and BR610 ("blue membrane", 4%). BR490 appears particularly in the additional fluorescence band at 550 nm and is assigned to the dehydrated species of bacteriorhodopsin. The presence of that particular species indicates the suppression of rehydration in a partially dehydrated crystal and shows that the three dimensional arrangement hinders water diffusion. A simple model, close domains are assumed to form, eventually suppressing free water diffusion in a crystal. The lack of water on the protein surface modifies the protonation states of the amino acids in the binding pocket and therefore the optical properties of the retinal. The chromophore actually reacts on the alteration of its distant environment upon incorporation in the protein crystal. The second distance scale - the fuzzy environment - is addressed by a time-integrated fluorescence spectroscopy on native bacteriorhodopsin as function of the excitation wavelength (430 nm to 650 nm). The optimized experimental set-up guaranteed that the fluorescence of only the excited state I460 contributed. The measured spectra show maxima at 740 nm, and exhibit a Stokes shift of 5000 cm-1, independent of the excitation wavelength. The similarity of the fluorescence excitation spectrum with the spectrum of absorbed photons excludes energy-dependent loss channels. However, the spectra show a growing asymmetric broadening on the high-energy side with decreasing excitation wavelength. The additional fluorescence originates from vibrational hot states, also supported by the decomposition by Gaussian functions. A comparison with the fluorescence spectrum of blue membranes shows that the low-energy part also arises from vibrational hot states. Consequently, the first fast intramolecular vibrational energy relaxation does not entirely distribute the access-energy to other modes. The remaining energy is found in a quasi-static occupation of vibrational modes. The subsequent second vibrational relaxation — probably not only involving the retinal but also the protein — takes place on a time scale similar to that of the isomerization. The fast isomerization reaction prevents dissipation of energy relaxation of the excited state of the retinal into the protein environment. On a third distance scale, femtosecond spectroscopy in the UV offers a direct view into the electrostatic interaction of the chromophore with its immediate neighborhood. For the first time, the retinal isomerization was studied within the spectral window of the tryptophan absorption from 265 nm to 310 nm. For this, a set-up adapted to UV wavelengths was realized, basing on non-linear optics. The developed single-shot detection system yields a sensitivity of 10-5 in optical density. In the transient absorption measurements, a time resolution of 90 fs was achieved. At short probe wavelengths (265 nm - 294 nm), the measured transients show first a fast reduction of the absorption which subsequently recovers on three time scales (380 fs, 3 ps, > 16 ps). With increasing probe wavelength an induced transient absorption becomes dominant (282 nm - 294 nm). It has a rise time of 280 fs, and decays with a time constant of 4.3 ps. With further increasing probe wavelength the absorption signal decays faster. In response to the complex signature of the transients, an iterative and model-independent transient-decomposition is introduced. A set of three basis elements were identified: one bleach, and two absorption transients. The two induced absorption transients are assigned to retinal. The dynamics of the excited state (I460) in the first retinal-transient is described by two time constants (280 fs and 1 ps). The fast time constant leads to the photoproduct, while the slower one leads back to the ground state, presumably by internal conversion. The 250 fs delayed onset of the second transient (photo-intermediate J) is connected to a wave packet motion in the excited state. This delayed onset of the transient was observed for the first time. It demands a rise time of the J photo-intermediate of only 280 fs. This contrasts common estimates of 500 fs given in literature. For the first time, the temporal behavior of the permanent dipole moment of retinal is observed experimentally. Its evolution is imprinted in the dominant bleach feature (265 nm - 294 nm), originating from tryptophan Trp86. In the excited state, the permanent dipole moment increases strongly within the first 250 fs, and decays exponentially afterwards, following the formation of the photoproduct. However, the related time constant of 380 fs is slightly greater than that of 280 fs of the photoproduct J. This discrepancy may indicate a remaining long-lived polarization of the binding pocket. In brief, by using the intrinsic tryptophan as a probe, the local evolution of the electric field during the isomerization reaction was investigated. Indeed the isomerization reaction is reflected in the immediate environment. The variety of optical spectroscopy provides methods especially adapted for studying the influences of the environment on a chemical reaction. The results achieved here highlight the importance of these influences.

EPFL2004