Matthew Tremblay

Ribonucleotide reductase (RNR) is an essential enzyme that converts ribonucleotides to deoxyribonucleotides, utilizing a ∼32 Å chain of proton-coupled electron transfer (PCET) reactions spanning two protein subunits to generate a catalytic cysteine radical. Two cryogenic electron microscopy structures of the active complex of E. coli RNR are currently available. One structure was trapped in the preturnover state, prior to radical translocation, and the other structure was trapped in the midturnover state, with the radical in the active site. Herein, we use molecular dynamics simulations to investigate the differences in hydrogen-bonding interactions and conformational motions between the preturnover and midturnover states. Our simulations show that Y731, an interfacial tyrosine that participates in the PCET pathway, samples multiple conformations in both states, allowing it to participate in forward and reverse PCET between subunits. We also observe interfacial water channels between the protein subunits in both states. Moreover, our simulations show that E623, which is near Y730 in the preturnover structure and was shown by previous simulations to mediate PCET between Y731 and Y730, can also sample conformations distal to Y730 in the preturnover state, similar to its position in the midturnover structure. Our mixed quantum mechanical/molecular mechanical free energy simulations indicate that forward radical transfer from Y731 to Y730 is thermodynamically favorable with a reasonable free energy barrier, even in the absence of mediation by E623. These results provide insights into the critical role of conformational motions and flexibility in regulating PCET reactions at and near the protein subunit interface of RNR.

Protein stabilizers and denaturants can be used to elucidate the fundamental principles of hydration, which is crucial for biological functions and biotechnology. Despite decades of work, existing molecular models of such stabilizers and denaturants have not yet been fully validated because few experimental methods can detect water structures within the hydration shell in situ under ambient conditions. Here, we devise a molecular model guided by the application of chiral-selective vibrational sum frequency generation spectroscopy to a solvated protein. We show that urea disrupts the first hydration shell, weakening the protein’s hydrogen bonds. Conversely, trimethylamine N-oxide resides outside the shell and stabilizes the protein by strengthening water hydrogen bonds within the first hydration shell. In mixtures, trimethylamine N-oxide drives urea out of the shell, neutralizing urea’s destabilizing effect. We conclude that protein stability directly correlates with first hydration shell integrity. These insights have broad implications for understanding solvent effects on biocatalysis and heterogeneous cellular environments.

Cryptochrome 4a (Cry4a) is a leading candidate for the radical pair-based magnetoreceptor proposed to enable avian navigation. Following photoexcitation, electron transfer along a tryptophan tetrad generates radical pairs whose recombination dynamics are thought to underlie magnetic sensitivity. However, competing proton-related reactions that may modulate these spin-selective processes remain poorly understood. Here, we combine classical molecular dynamics and quantum mechanical/molecular mechanical free energy simulations to investigate deprotonation of terminal tryptophan radical cations and a potential proton-coupled electron transfer (PCET) pathway involving a surface-exposed tyrosine. We find that limited solvent accessibility of the third tryptophan significantly suppresses its effective deprotonation, whereas the fourth tryptophan is more readily deprotonated despite similar intrinsic proton transfer thermodynamics. In addition, we identify a multisite PCET pathway in which electron transfer from tyrosine to the fourth tryptophan radical cation is coupled to proton transfer from tyrosine to interfacial water, with a free energy barrier consistent with sub-microsecond kinetics. These results demonstrate that proton transfer and PCET reactions at the protein-solvent interface can compete kinetically with radical pair recombination, thereby providing alternative pathways that may influence magnetic sensitivity. This work establishes a mechanistic framework for probing proton-coupled processes in cryptochrome-based magnetoreception.

Investigating water structures at aqueous interfaces has been a central focus in the field of nonlinear surface spectroscopy over decades. This large body of work leads to a conclusion that asymmetric OH stretches should be largely silent in sum frequency generation (SFG) spectra. Nonetheless, our recent studies show chiral-specific SFG response of water originating from the first hydration shell of proteins and DNA arising from a sum of equal-magnitude and opposite-phase symmetric and asymmetric OH stretches, resulting in an oppositely signed couplet. Motivated initially by this apparently inconsistent behavior for chiral-specific vs conventional (achiral) SFG, we demonstrate herein a fundamental argument for the appearance of the oppositely signed couplets in chiral-specific vibrational SFG. The interplay between two foundational optical relations, Kramers–Kronig relations and index-interchange symmetry arising in the adiabatic zero-frequency limit (often referred to as Kleinman symmetry), is shown to require the imaginary resonant contributions to the tensors describing chiral-specific SFG responses to collectively sum to zero. In brief, all indices within the surface susceptibility must become interchangeable in the degenerate, zero-frequency adiabatic limit. From Kramers–Kronig relations connecting the real and imaginary susceptibility, this asymptotic limit in the real susceptibility can only be met if the imaginary-valued resonant contributions over the relevant spectral range sum to zero. These symmetry constraints were found to agree with density functional theory calculations on small water clusters and with experimental and computational phase-resolved chiral-specific SFG spectra. These symmetry requirements provide constraints for analyzing phase-resolved chiral-specific SFG spectra for extracting structural information about chiral molecules at interfaces.

Investigating water structures at aqueous interfaces has been a central focus in the field of nonlinear surface spectroscopy over decades. This large body of work leads to a conclusion that asymmetric OH stretches should be largely silent in sum frequency generation (SFG) spectra. Nonetheless, our recent studies show chiral-specific SFG response of water originated from the first hydration shell of proteins and DNA arising from a sum of equal-magnitude and opposite- phase symmetric and asymmetric OH stretches, resulting in an oppositely-signed couplet. Motivated initially by this apparently inconsistent behavior for chiral-specific versus conventional (achiral) SFG, we demonstrate herein a fundamental argument for the appearance of the oppositely-signed couplets in chiral-specific vibrational SFG. The interplay between two foundational optical relations, Kramers-Kronig relations and Kleinman symmetry, is shown to require the imaginary resonant contributions to the tensors describing chiral-specific SFG responses to collectively sum to zero. In brief, all indices within the surface susceptibility must become interchangeable in the degenerate, zero-frequency limit. From Kramers-Kronig relations connecting the real and imaginary susceptibility, this asymptotic limit in the real susceptibility can only be met if the imaginary-valued resonant contributions over the relevant spectral range sum to zero. These symmetry constraints were found to agree with density functional theory calculations on small water clusters and with experimental and computational phase-resolved chiral-specific SFG spectra. These symmetry requirements provide constraints for analyzing phase-resolved chiral-specific SFG spectra for extracting structural information about chiral molecules at interfaces.

Knowledge of how intermolecular interactions change hydration structures surrounding DNA will heighten understanding of DNA biology and advance drug development. However, probing changes in DNA hydration structures in response to molecular interactions and drug binding in situ under ambient conditions has remained challenging. Here, we apply a combined experimental and computational approach of chiral-selective vibrational sum frequency generation spectroscopy (chiral SFG) to probe changes of DNA hydration structures when a small-molecule drug, netropsin, binds the minor groove of DNA. Our results show that chiral SFG can detect water being displaced from the minor groove of DNA due to netropsin binding. Additionally, we observe that chiral SFG distinguishes between weakly and strongly hydrogen-bonded water hydrating DNA. Chiral SFG spectra show that netropsin binding, instead of displacing weakly hydrogen-bonded water, preferentially displaces water molecules strongly hydrogen-bonded to thymine carbonyl groups in the DNA minor groove, revealing the roles of water in modulating site-specificity of netropsin binding to duplex DNA rich in adenine–thymine sequences. The results convey the promise of chiral SFG to offer mechanistic insights into roles of water in drug development targeting DNA.

Knowledge of how intermolecular interactions change hydration structures surrounding DNA will heighten understanding of DNA biology and advance drug development. However, probing changes in DNA hydration structures in response to molecular interactions and drug binding in situ under ambient conditions has remained challenging. Here, we apply a combined experimental and computational approach of chiral-selective vibrational sum frequency generation spectroscopy (chiral SFG) to probe changes of DNA hydration structures when a small-molecule drug, netropsin, binds the minor groove of DNA. Our results show that chiral SFG can detect water being displaced from the minor groove of DNA due to netropsin binding. Additionally, we observe that chiral SFG distinguishes between weakly and strongly hydrogen-bonded water hydrating DNA. Chiral SFG spectra show that netropsin binding, instead of displacing weakly hydrogen-bonded water, preferentially displaces water molecules strongly hydrogen-bonded to thymine carbonyl groups in the DNA minor groove, revealing the roles of water in modulating site-specificity of netropsin binding to duplex DNA rich in adenine-thymine sequences. The results convey the promise of chiral SFG to offer mechanistic insights into roles of water in drug development targeting DNA.

Experimental methods capable of selectively probing water at the DNA minor groove, major groove, and phosphate backbone are crucial for understanding how hydration influences DNA structure and function. Chiral-selective sum frequency generation spectroscopy (chiral SFG) is unique among vibrational spectroscopies because it can selectively probe water molecules that form chiral hydration structures around biomolecules. However, interpreting chiral SFG spectra is challenging since both water and the biomolecule can produce chiral SFG signals. Here, we combine experiment and computation to establish a theoretical framework for the rigorous interpretation of chiral SFG spectra of DNA. We demonstrate that chiral SFG detects the N–H stretch of DNA base pairs and the O–H stretch of water, exclusively probing water molecules in the DNA first hydration shell. Our analysis reveals that DNA transfers chirality to water molecules only within the first hydration shell, so they can be probed by chiral SFG spectroscopy. Beyond the first hydration shell, the electric field-induced water structure is symmetric and, therefore, precludes chiral SFG response. Furthermore, we find that chiral SFG can differentiate chiral subpopulations of first hydration shell water molecules at the minor groove, major groove, and phosphate backbone. Our findings challenge the scientific perspective dominant for more than 40 years that the minor groove “spine of hydration” is the only chiral water structure surrounding the DNA double helix. By identifying the molecular origins of the DNA chiral SFG spectrum, we lay a robust experimental and theoretical foundation for applying chiral SFG to explore the chemical and biological physics of DNA hydration.

Experimental methods capable of selectively probing water at the DNA minor groove, major groove, and phosphate backbone are crucial for understanding how hydration influences DNA structure and function. Chiral-selective sum frequency generation spectroscopy (chiral SFG) is unique among vibrational spectroscopies because it can selectively probe water molecules that form chiral hydration structures around biomolecules. However, interpreting chiral SFG spectra is challenging since both water and the biomolecule can produce chiral SFG signals. Here, we combine experiment and computation to establish a theoretical framework for rigorous interpretation of chiral SFG spectra of DNA. We demonstrate that chiral SFG detects the N-H stretch of DNA base pairs and the O-H stretch of water, exclusively probing water molecules in the DNA first hydration shell. Our analysis reveals that DNA transfers chirality to water molecules only within the first hydration shell, so they can be probed by chiral SFG spectroscopy. Beyond the first hydration shell, electric field-induced water structure is symmetric and therefore precludes chiral SFG response. Furthermore, we find that chiral SFG can differentiate chiral subpopulations of first hydration shell water molecules at the minor groove, major groove, and phosphate backbone. Our findings challenge the scientific perspective dominant for more than 40 years that the minor groove “spine of hydration” is the only chiral water structure surrounding the DNA double helix. By identifying the molecular origins of the DNA chiral SFG spectrum, we lay a robust experimental and theoretical foundation for applying chiral SFG to explore the chemical and biological physics of DNA hydration.

Chirality-selective vibrational sum frequency generation (chiral SFG) spectroscopy has emerged as a powerful technique for the study of biomolecular hydration water due to its sensitivity to the induced chirality of the first hydration shell. Thus far, water O–H vibrational bands in phase-resolved heterodyne chiral SFG spectra have been fit using one Lorentzian function per vibrational band, and the resulting fit has been used to infer the underlying frequency distribution. Here, we show that this approach may not correctly reveal the structure and dynamics of hydration water. Our analysis illustrates that the chiral SFG responses of symmetric and asymmetric O–H stretch modes of water have opposite phase and equal magnitude and are separated in energy by intramolecular vibrational coupling and a heterogeneous environment. The sum of the symmetric and asymmetric responses implies that an O–H stretch in a heterodyne chiral SFG spectrum should appear as two peaks with opposite phase and equal amplitude. Using pairs of Lorentzian functions to fit water O–H stretch vibrational bands, we improve spectral fitting of previously acquired experimental spectra of model β-sheet proteins and reduce the number of free parameters. The fitting allows us to estimate the vibrational frequency distribution and thus reveals the molecular interactions of water in hydration shells of biomolecules directly from chiral SFG spectra.