Mark Mero

PAS domains mediate protein-protein interactions that enable functions such as sensing, signaling, dimerization, and localization. The photoactive yellow protein (PYP) from Halorhodospira halophila is a model PAS-domain protein involved in negative phototaxis, yet its signaling partner remains unidentified. Here, we present a method to resolve protein orientations in PAS-domain signaling by combining nano-FTIR and chiral vibrational sum-frequency generation (VSFG) spectroscopy with molecular dynamics simulations and VSFG spectral calculations. As a demonstration, we used a charged homopolypeptide, poly-l-lysine (PLL), as a surrogate binding surface to probe PYP docking. We found that PYP adopts a preferred interfacial orientation driven primarily by dipole-dipole interactions, despite its water-soluble, i.e., cytoplasmic nature. Remarkably, the inferred interaction surface and orientation closely match those observed in PYP homodimers and in a CNBh-PAS heterodimer. This methodology enables in situ determination of protein orientational preferences during protein-protein interactions and may facilitate identification of binding partners in PAS-domain signaling pathways.

Chiral VSFG and nano-FTIR data show that protein adsorption is governed not only by electrostatics but also by the secondary-structure architecture of the underlying scaffold, which controls monolayer formation, molecular alignment, and hydration.

We investigate the nonlinear optical response of bulk ZnO under intense short-wave infrared excitation, focusing on the interplay between high-harmonic generation (HHG) and photoluminescence (PL). While HHG exhibits nonperturbative intensity scaling and a spectral blueshift consistent with plasma-induced refractive index changes, the PL signal shows a pronounced superlinear increase and a redshift, attributed to a combination of exciton-exciton scattering and phonon-assisted exciton recombination emission. A similar PL response under above-bandgap excitation supports its intrinsic origin. Spectral analysis of the HHG emission reveals an intensity-driven transition in the characteristics of the fifth harmonic, indicating a change in the underlying generation mechanism. These findings establish PL and spectral HHG analysis as complementary probes of strong-field and many-body effects in wide-bandgap semiconductors.

We report on a high-energy Ho:YLF chirped pulse amplifier (CPA) operating at 1 kHz. The amplifier chain is composed of a regenerative amplifier (RA) and a power amplifier containing two stages. The 25-kHz seed source is an optical parametric chirped pulse amplifier based on a Yb:KGW laser. It delivers 44 µJ pulses centered at 2050 nm with a duration of 114 fs, of which 4 µJ can be utilized for seeding the Ho:YLF RA. Profiting from the high seed energy, gain narrowing is reduced and bifurcation instabilities are attenuated in the high-gain RA. The Ho:YLF RA generated pulses with 22 mJ energy in the saturated single-pulse regime. The pulse energy is scaled-up in two consecutive single-pass power amplifiers to 75 mJ corresponding to an average power of 75 W. The subsequent compression of the pulses results in 2.2 ps duration, corresponding to a peak power of 31 GW. The water cooled Ho:YLF amplifier chain is characterized by an excellent stability of 0.19% rms and a remarkable extraction efficiency of 22%. The demonstrated pulse energy and peak power are record values for few-ps CPAs operating near 2 µm wavelength.

Moving the detection wavelength in vibrational sum-frequency generation (VSFG) spectroscopy to the near-infrared (> 700 nm) is crucial to avoid autofluorescence and electronic resonance effects distorting the molecular phase response or triggering unwanted chemical and photophysical reactions. Here, we employ an ultra-narrow bandpass interference filter on Yb laser pulses to generate an upconversion beam near 1.03 µm at a spectral resolution of 5 cm^-1. We demonstrate that the beam rejected by the filter can be utilized for driving a supercontinuum-seeded near-infrared optical parametric amplifier, boosting the seed energy for a broadband LiGaS₂-based mid-infrared amplifier. Through the example of a chromoprotein, we show that utilizing the scheme can extend the range of interfacial molecular systems that can be studied using VSFG spectroscopy, even in the case of high repetition rate Yb pump lasers with limited pulse energy. The scheme can also be utilized in other types of vibrational spectroscopies that derive their spectral resolution from bandpass-filtering of femtosecond near-infrared laser pulses, such as stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) spectroscopy.

Ultrafast electron diffraction (UED) instruments typically operate at kHz or lower repetition rates and rely on indirect detection of electrons. However, these experiments encounter limitations because they are required to use electron beams containing a relatively large number of electrons (≫100 electrons/pulse), leading to severe space-charge effects. Consequently, electron pulses with long durations and large transverse diameters are used to interrogate the sample. Here, we introduce a novel UED instrument operating at a high repetition rate and employing direct electron detection. We operate significantly below the severe space-charge regime by using electron beams containing 1–140 electrons per pulse at 30 kHz. We demonstrate the ability to detect time-resolved signals from thin film solid samples with a difference contrast signal, ΔI/I0, and an instrument response function as low as 10−5 and 184-fs (FWHM), respectively, without temporal compression. Overall, our findings underscore the importance of increasing the repetition rate of UED experiments and adopting a direct electron detection scheme, which will be particularly impactful for gas-phase UED. Our newly developed scheme enables more efficient and sensitive investigations of ultrafast dynamics in photoexcited samples using ultrashort electron beams.

We report on a high-energy, few-ps, continuous-wave pumped Ho:YLF regenerative amplifier (RA) operating in a chirped-pulse amplification arrangement. A three-stage optical parametric amplifier serves as versatile seed source emitting broadband pulses centered at 2050 nm. It provides seed pulses with 4 µJ energy within the Ho:YLF amplification bandwidth centered at 2051 nm. Thanks to the high seed pulse energy, bifurcation instabilities are mitigated and gain narrowing is reduced for such a high-gain Ho-doped RA operating at few kHz repetition rate. At 1 kHz the Ho:YLF RA emits 22.5 mJ energy pulses with a compressed pulse duration of 2.0 ps in the stable single-pulse regime. The water cooled RA features a high extraction efficiency of 31% and exhibits a remarkable stability of 0.11% rms. Doubling the repetition rate to 2 kHz, RA pulse energy reaches 15 mJ. The demonstrated 10 GW peak power at 1 kHz and the 30 W average power at 2 kHz are the highest values reported to date for few-ps RAs operating at 2 µm wavelength.

PAS domains mediate protein-protein interactions, which give them key functions ranging from sensing and signaling to dimerization and localization. PYP is one of the PAS-domain model proteins that regulates negative phototactic signal response, and its photoreaction is well characterized. In turn, a protein dimer is usually required to maintain its function as a PAS family domain. However, no partner protein for the signaling pathway of PYP has been identified yet. Here, we investigated the intermolecular interaction of PYP with a charged homopolypeptide, poly-L-lysine, in situ by combining chiral vibrational sum-frequency generation (VSFG) spectroscopy with molecular dynamics simulations and VSFG spectral calculations. Our combined experimental and theoretical approach enabled us to study the structure and orientation of proteins at interfaces based on probing the chiral N-H stretching and amide I and II vibrational modes. We found that PYP has a preferred orientation at the PLL interface, mainly due to dipole-dipole interaction, despite its water-soluble, i.e., cytoplasmic nature. Interestingly, the interaction surface and the orientation of PYP resulted in our model being nearly identical to a homodimer of PYP and a heterodimer in CNBh domains consisting of a PAS member. Our methodology provides a promising route for revealing orientational preferences of molecular groups during in situ protein-protein interactions and may ultimately help identify target proteins in PAS-domain signaling pathways.

Chiral and achiral vibrational sum-frequency generation (VSFG) spectroscopy was performed in the 1400–1700 and 2800–3800 cm⁻¹ range to study the interfacial structure of photoactive yellow protein (PYP) adsorbed on polyethyleneimine (PEI) and poly-l-glutamic acid (PGA) surfaces. Nanometer-thick polyelectrolyte layers served as the substrate for PYP adsorption, with 6.5-pair layers providing the most homogeneous surfaces. When the topmost material was PGA, it acquired a random coil structure with a small number of β₂-fibrils. Upon adsorption on oppositely charged surfaces, PYP yielded similar achiral spectra. However, the VSFG signal intensity increased for PGA surfaces with a concomitant redshift of the chiral C^α-H and N–H stretching bands, suggesting increased adsorption for PGA compared to PEI. At low wavenumbers, both the backbone and the side chains of PYP induced drastic changes to all measured chiral and achiral VSFG spectra. Decreasing ambient humidity led to the loss of tertiary structure with a re-orientation of α-helixes, evidenced by a strongly blue-shifted chiral amide I band of the β-sheet structure with a shoulder at 1654 cm⁻¹. Our observations indicate that chiral VSFG spectroscopy is not only capable of determining the main type of secondary structure of PYP, i.e., β-scaffold, but is also sensitive to tertiary protein structure.