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In this work we present a high-throughput approach to the computation of absorption UV-Vis spectra tailored to mutagenesis studies. The scheme makes use of a single molecular dynamics trajectory of a reference (non-mutated) species. The shifts in absorption energy caused by a residue mutation are evaluated by building an effective potential of the environment and computing a correction term based on perturbation theory. The sampling is only performed in the phase space of the initial protein. We analyze the robustness of the method by comparing different approximations for the effective potential, the sampling of mutant residue geometries and observing the impact in the prediction of both bathocromic and hypsochromic shifts. As a test subject, we consider a red fluorescent protein variant with potential biotechnological applications.

Electronic absorption spectra are oftentimes used to identify reaction intermediates or substrates/products in enzymatic systems, as long as absorption bands can be unequivocally assigned to the species being studied. The latter task is far from trivial given the transient nature of some states and the complexity of the surrounding environment around the active site. To identify unique spectral fingerprints, controlled experiments with model compounds have been used in the past, but even these can sometimes be unreliable. Circular dichroism (CD) and ultraviolet-visible spectra have been tools of choice in the study of the rich chemistry of thiamin diphosphate-dependent enzymes. In this study, we focus on the Zymomonas mobilis pyruvate decarboxylase, and mutant analogues thereof, as a prototypical representative of the thiamin diphosphate (ThDP) enzyme superfamily. Through the use of electronic structure methods, we analyze the nature of electronic excitations in the cofactor. We find that all the determining CD bands around the 280-340 nm spectral range correspond to charge-transfer excitations between the pyrimidine and thiazolium rings of ThDP, which, most likely, is a general property of related ThDP-dependent enzymes. While we can confirm the assignments of previously proposed bands to chemical states, our calculations further suggest that a hitherto unassigned band of enzyme-bound ThDP reports on the ionization state of the canonical glutamate that is required for cofactor activation. This finding expands the spectroscopic "library" of chemical states of ThDP enzymes, permitting a simultaneous assignment of both the cofactor ThDP and the activating glutamate. We anticipate this finding to be helpful for mechanistic analyses of related ThDP enzymes.

The underlying molecular mechanisms of cooperativity and allosteric regulation are well understood for many proteins, with haemoglobin and aspartate transcarbamoylase serving as prototypical examples1,2. The binding of effectors typically causes a structural transition of the protein that is propagated through signalling pathways to remote sites and involves marked changes on the tertiary and sometimes even the quaternary level1-5. However, the origin of these signals and the molecular mechanism of long-range signalling at an atomic level remain unclear5-8. The different spatial scales and timescales in signalling pathways render experimental observation challenging; in particular, the positions and movement of mobile protons cannot be visualized by current methods of structural analysis. Here we report the experimental observation of fluctuating low-barrier hydrogen bonds as switching elements in cooperativity pathways of multimeric enzymes. We have observed these low-barrier hydrogen bonds in ultra-high-resolution X-ray crystallographic structures of two multimeric enzymes, and have validated their assignment using computational calculations. Catalytic events at the active sites switch between low-barrier hydrogen bonds and ordinary hydrogen bonds in a circuit that consists of acidic side chains and water molecules, transmitting a signal through the collective repositioning of protons by behaving as an atomistic Newton's cradle. The resulting communication synchronizes catalysis in the oligomer. Our studies provide several lines of evidence and a working model for not only the existence of low-barrier hydrogen bonds in proteins, but also a connection to enzyme cooperativity. This finding suggests new principles of drug and enzyme design, in which sequences of residues can be purposefully included to enable long-range communication and thus the regulation of engineered biomolecules.