Accurate analysis of HCl in biomethane using laser absorption spectroscopy and ion-exchange chromatography.

Biomethane is a renewable energy gas with great potential to contribute to the diversification and greening of the natural gas supply. Ideally, biomethane can directly be injected into the natural gas grid system. For grid injection, specifications such as those in EN 16723-1 shall be met. One of the impurities to be monitored is hydrogen chloride (HCl). To assess conformity with the specification for HCl, accurate and reliable test methods are required. Here, we report the development of three novel test methods, based on a variety of laser absorption spectroscopy techniques (Direct absorption spectroscopy-DAS and wavelength modulation spectroscopy-WMS) and ion-exchange chromatography, for the measurement of HCl in biomethane. Gas mixtures of HCl in biomethane were used to demonstrate the performance of the spectroscopic systems in the nmol mol-1 to low µmol mol-1 ranges, achieving uncertainties in the 4% range, k = 2. For ion-exchange chromatography analysis, HCl was first collected on an alkali-impregnated quartz fiber filter. The analysis was performed according to ISO 21438-2 and validated using synthetic biomethane spiked with HCl. The relative expanded uncertainties for the ion exchange chromatography HCl measurements are in the 10-37% range, k = 2. The results presented for the 3 test methods demonstrate that the respective methods can be used for HCl conformity assessment in biomethane.

Folding of a Zinc-Finger ßßα-Motif Investigated Using Two-Dimensional and Time-Resolved Vibrational Spectroscopy.

Small proteins provide good model systems for studying the fundamental forces that control protein folding. Here, we investigate the folding dynamics of the 28-residue zinc-finger mutant FSD-1, which is designed to form a metal-independent folded ßßα-motif, and which provides a testing ground for proteins containing a mixed α/ß fold. Although the folding of FSD-1 has been actively studied, the folding mechanism remains largely unclear. In particular, it is unclear in what stage of folding the α-helix is formed. To address this issue we investigate the folding mechanism of FSD-1 using a combination of temperature-dependent UV circular dichroism (UV-CD), Fourier transform infrared (FTIR) spectroscopy, two-dimensional infrared (2D-IR) spectroscopy, and temperature-jump (T-jump) transient-IR spectroscopy. Our UV-CD and FTIR data show different thermal melting transitions, indicating multistate folding behavior. Temperature-dependent 2D-IR spectra indicate that the α-helix is the most stable structural element of FSD-1. To investigate the folding/unfolding re-equilibration dynamics of FSD-1, the conformational changes induced by a nanosecond T-jump are probed with transient-IR and transient dispersed-pump-probe (DPP) IR spectroscopy. We observe biexponential T-jump relaxation kinetics (with time constants of 80 ± 13 ns and 1300 ± 100 ns at 322 K), confirming that the folding involves an intermediate state. The IR and dispersed-pump-probe IR spectra associated with the two kinetic components suggest that the folding of FSD-1 involves early formation of the α-helix, followed by the formation of the ß-hairpin and hydrophobic contacts.

Influence of Glu/Arg, Asp/Arg, and Glu/Lys Salt Bridges on α-Helical Stability and Folding Kinetics.

Using a combination of ultraviolet circular dichroism, temperature-jump transient-infrared spectroscopy, and molecular dynamics simulations, we investigate the effect of salt bridges between different types of charged amino-acid residue pairs on α-helix folding. We determine the stability and the folding and unfolding rates of 12 alanine-based α-helical peptides, each of which has a nearly identical composition containing three pairs of positively and negatively charged residues (either Glu(-)/Arg(+), Asp(-)/Arg(+), or Glu(-)/Lys(+)). Within each set of peptides, the distance and order of the oppositely charged residues in the peptide sequence differ, such that they have different capabilities of forming salt bridges. Our results indicate that stabilizing salt bridges (in which the interacting residues are spaced and ordered such that they favor helix formation) speed up α-helix formation by up to 50% and slow down the unfolding of the α-helix, whereas salt bridges with an unfavorable geometry have the opposite effect. Comparing the peptides with different types of charge pairs, we observe that salt bridges between side chains of Glu(-) and Arg(+) are most favorable for the speed of folding, probably because of the larger conformational space of the salt-bridging Glu(-)/Arg(+) rotamer pairs compared to Asp(-)/Arg(+) and Glu(-)/Lys(+). We speculate that the observed impact of salt bridges on the folding kinetics might explain why some proteins contain salt bridges that do not stabilize the final, folded conformation.

Guanidinium-Induced Denaturation by Breaking of Salt Bridges.

Despite its wide use as a denaturant, the mechanism by which guanidinium (Gdm(+) ) induces protein unfolding remains largely unclear. Herein, we show evidence that Gdm(+) can induce denaturation by disrupting salt bridges that stabilize the folded conformation. We study the Gdm(+) -induced denaturation of a series of peptides containing Arg/Glu and Lys/Glu salt bridges that either stabilize or destabilize the folded conformation. The peptides containing stabilizing salt bridges are found to be denatured much more efficiently by Gdm(+) than the peptides containing destabilizing salt bridges. Complementary 2D-infrared measurements suggest a denaturation mechanism in which Gdm(+) binds to side-chain carboxylate groups involved in salt bridges.

The structure of salt bridges between Arg(+) and Glu(-) in peptides investigated with 2D-IR spectroscopy: Evidence for two distinct hydrogen-bond geometries.

Salt bridges play an important role in protein folding and in supramolecular chemistry, but they are difficult to detect and characterize in solution. Here, we investigate salt bridges between glutamate (Glu(-)) and arginine (Arg(+)) using two-dimensional infrared (2D-IR) spectroscopy. The 2D-IR spectrum of a salt-bridged dimer shows cross peaks between the vibrational modes of Glu(-) and Arg(+), which provide a sensitive structural probe of Glu(-)⋯Arg(+) salt bridges. We use this probe to investigate a ß-turn locked by a salt bridge, an α-helical peptide whose structure is stabilized by salt bridges, and a coiled coil that is stabilized by intra- and intermolecular salt bridges. We detect a bidentate salt bridge in the ß-turn, a monodentate one in the α-helical peptide, and both salt-bridge geometries in the coiled coil. To our knowledge, this is the first time 2D-IR has been used to probe tertiary side chain interactions in peptides, and our results show that 2D-IR spectroscopy is a powerful method for investigating salt bridges in solution.

Communication: nanosecond folding dynamics of an alpha helix: time-dependent 2D-IR cross peaks observed using polarization-sensitive dispersed pump-probe spectroscopy.

We present a simple method to measure the dynamics of cross peaks in time-resolved two-dimensional vibrational spectroscopy. By combining suitably weighted dispersed pump-probe spectra, we eliminate the diagonal contribution to the 2D-IR response, so that the dispersed pump-probe signal contains the projection of only the cross peaks onto one of the axes of the 2D-IR spectrum. We apply the method to investigate the folding dynamics of an alpha-helical peptide in a temperature-jump experiment and find characteristic folding and unfolding time constants of 260 ± 30 and 580 ± 70 ns at 298 K.

Solvent-Exposed Salt Bridges Influence the Kinetics of α-Helix Folding and Unfolding.

Salt bridges are known to play an essential role in the thermodynamic stability of the folded conformation of many proteins, but their influence on the kinetics of folding remains largely unknown. Here, we investigate the effect of Glu-Arg salt bridges on the kinetics of α-helix folding using temperature-jump transient-infrared spectroscopy and steady-state UV circular dichroism. We find that geometrically optimized salt bridges (Glu- and Arg+ are spaced four peptide units apart, and the Glu/Arg order is such that the side-chain rotameric preferences favor salt-bridge formation) significantly speed up folding and slow down unfolding, whereas salt bridges with unfavorable geometry slow down folding and slightly speed up unfolding. Our observations suggest a possible explanation for the surprising fact that many biologically active proteins contain salt bridges that do not stabilize the native conformation: these salt bridges might have a kinetic rather than a thermodynamic function.

Folding dynamics of the Trp-cage miniprotein: evidence for a native-like intermediate from combined time-resolved vibrational spectroscopy and molecular dynamics simulations.

Trp-cage is a synthetic 20-residue miniprotein which folds rapidly and spontaneously to a well-defined globular structure more typical of larger proteins. Due to its small size and fast folding, it is an ideal model system for experimental and theoretical investigations of protein folding mechanisms. However, Trp-cage's exact folding mechanism is still a matter of debate. Here we investigate Trp-cage's relaxation dynamics in the amide I' spectral region (1530-1700 cm(-1)) using time-resolved infrared spectroscopy. Residue-specific information was obtained by incorporating an isotopic label ((13)C═(18)O) into the amide carbonyl group of residue Gly11, thereby spectrally isolating an individual 310-helical residue. The folding-unfolding equilibrium is perturbed using a nanosecond temperature-jump (T-jump), and the subsequent re-equilibration is probed by observing the time-dependent vibrational response in the amide I' region. We observe bimodal relaxation kinetics with time constants of 100 ± 10 and 770 ± 40 ns at 322 K, suggesting that the folding involves an intermediate state, the character of which can be determined from the time- and frequency-resolved data. We find that the relaxation dynamics close to the melting temperature involve fast fluctuations in the polyproline II region, whereas the slower process can be attributed to conformational rearrangements due to the global (un)folding transition of the protein. Combined analysis of our T-jump data and molecular dynamics simulations indicates that the formation of a well-defined α-helix precedes the rapid formation of the hydrophobic cage structure, implying a native-like folding intermediate, that mainly differs from the folded conformation in the orientation of the C-terminal polyproline II helix relative to the N-terminal part of the backbone. We find that the main free-energy barrier is positioned between the folding intermediate and the unfolded state ensemble, and that it involves the formation of the α-helix, the 310-helix, and the Asp9-Arg16 salt bridge. Our results suggest that at low temperature (T ≪ Tm) a folding path via formation of α-helical contacts followed by hydrophobic clustering becomes more important.

Picosecond Raman spectroscopy with a fast intensified CCD camera for depth analysis of diffusely scattering media.

A spectroscopic depth profiling approach is demonstrated for layers of non-transparent, diffusely scattering materials. The technique is based on the temporal discrimination between Raman photons emitted from the surface and Raman photons originating from a deeper layer. Excitation was carried out with a frequency-doubled, 3 ps Ti:sapphire laser system (398 nm; 76 MHz repetition rate). Time-resolved detection was carried out with an intensified CCD camera that can be gated with a 250 ps gate width. The performance of the system was assessed using 1 mm and 2 mm pathlength cuvettes with powdered PMMA and trans-stilbene (TS) crystals, respectively, or solid white polymer blocks: Arnite (polyethylene terephthalate), Delrin (polyoxymethylene), polythene (polyethylene) and Teflon (polytetrafluoroethylene). These samples were pressed together in different configurations and Raman photons were collected in backscatter mode in order to study the time difference in such media corresponding with several mm of extra net photon migration distance. We also studied the lateral contrast between two different second layers. The results demonstrate that by means of a picosecond laser system and the time discrimination of a gated intensified CCD camera, molecular spectroscopic information can be obtained through a turbid surface layer. In the case of the PMMA/TS two-layer system, time-resolved detection with a 400 ps delay improved the relative intensity of the Raman bands of the second layer with a factor of 124 in comparison with the spectrum recorded with a 100 ps delay (which is more selective for the first layer) and with a factor of 14 in comparison with a non-gated setup. Possible applications will be discussed, as well as advantages/disadvantages over other Raman techniques for diffusely scattering media.