Ab initio chemical shielding calculations greatly facilitate the interpretation of nuclear magnetic resonance (NMR) chemical shifts in biological systems, but the large sizes of these systems requires approximations in the chemical models used to represent them. Achieving good convergence in the predicted chemical shieldings is necessary before one can unravel how other complex structural and dynamical factors affect the NMR measurements. Here, we investigate how to balance trade-offs between using a better basis set or a larger cluster model for predicting the chemical shieldings of the substrates in two representative examples of protein-substrate systems involving different domains in tryptophan synthase: the N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F9) ligand which binds in the α active site, and the 2-aminophenol quinonoid intermediate formed in the β active site. We first demonstrate that a chemically intuitive three-layer, locally dense basis model that uses a large basis on the substrate, a medium triple-zeta basis to describe its hydrogen-bonding partners and/or surrounding van der Waals cavity, and a crude basis set for more distant atoms provides chemical shieldings in good agreement with much more expensive large basis calculations. Second, long-range quantum mechanical interactions are important, and one can accurately estimate them as a small-basis correction to larger-basis calculations on a smaller cluster. The combination of these approaches enables one to perform density functional theory NMR chemical shift calculations in protein systems that are well-converged with respect to both basis set and cluster size.

What controls species diversity and diversification is one of the major questions in evolutionary biology and paleontology. Previous studies have addressed this issue based on various plant and animal groups, geographic regions, and time intervals. However, as most previous research focused on terrestrial or marine ecosystems, our understanding of the controls on diversification of biota (and particularly invertebrates) in freshwater environments in deep time is still limited. Here, we infer diversification rates of North American freshwater gastropods from the Late Triassic to the Pleistocene and explore potential links between shifts in speciation and extinction and major changes in paleogeography, climate, and biotic interactions. We found that variation in the speciation rate is best explained by changes in continental fragmentation, with rate shifts coinciding with major paleogeographic reorganizations in the Mesozoic, in particular the retreat of the Sundance Sea and subsequent development of the Bighorn wetland and the advance of the Western Interior Seaway. Climatic events in the Cenozoic (Middle Eocene Climate Optimum, Miocene Climate Optimum) variably coincide with shifts in speciation and extinction as well, but no significant long-term association could be detected. Similarly, no influence of diversity dependence was found across the entire time frame of ~ 214 Myr. Our results indicate that short-term climatic events and paleogeographic changes are relevant to the diversification of continental freshwater biota, while long-term trends have limited effect.

The allosteric regulation of substrate channeling in tryptophan synthase involves ligand-mediated allosteric signaling that switches the α- and β-subunits between open (low activity) and closed (high activity) conformations. This switching prevents the escape of the common intermediate, indole, and synchronizes the α- and β-catalytic cycles. (19)F NMR studies of bound α-site substrate analogues, N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), were found to be sensitive NMR probes of β-subunit conformation. Both the internal and external aldimine F6 complexes gave a single bound peak at the same chemical shift, while α-aminoacrylate and quinonoid F6 complexes all gave a different bound peak shifted by +1.07 ppm. The F9 complexes exhibited similar behavior, but with a corresponding shift of -0.12 ppm. X-ray crystal structures show the F6 and F9 CF3 groups located at the α-β subunit interface and report changes in both the ligand conformation and the surrounding protein microenvironment. Ab initio computational modeling suggests that the change in (19)F chemical shift results primarily from changes in the α-site ligand conformation. Structures of α-aminoacrylate F6 and F9 complexes and quinonoid F6 and F9 complexes show the α- and β-subunits have closed conformations wherein access of ligands into the α- and β-sites from solution is blocked. Internal and external aldimine structures show the α- and β-subunits with closed and open global conformations, respectively. These results establish that β-subunits exist in two global conformational states, designated open, where the β-sites are freely accessible to substrates, and closed, where the β-site portal into solution is blocked. Switching between these conformations is critically important for the αβ-catalytic cycle.

The acid-base chemistry that drives catalysis in pyridoxal-5'-phosphate (PLP)-dependent enzymes has been the subject of intense interest and investigation since the initial identification of PLP's role as a coenzyme in this extensive class of enzymes. It was first proposed over 50 years ago that the initial step in the catalytic cycle is facilitated by a protonated Schiff base form of the holoenzyme in which the linking lysine ε-imine nitrogen, which covalently binds the coenzyme, is protonated. Here we provide the first (15)N NMR chemical shift measurements of such a Schiff base linkage in the resting holoenzyme form, the internal aldimine state of tryptophan synthase. Double-resonance experiments confirm the assignment of the Schiff base nitrogen, and additional (13)C, (15)N, and (31)P chemical shift measurements of sites on the PLP coenzyme allow a detailed model of coenzyme protonation states to be established.

The scarcity of high-resolution empirical data directly tracking diversity over time limits our understanding of speciation and extinction dynamics and the drivers of rate changes. Here, we analyze a continuous species-level fossil record of endemic diatoms from ancient Lake Ohrid, along with environmental and climate indicator time series since lake formation 1.36 million years (Ma) ago. We show that speciation and extinction rates nearly simultaneously decreased in the environmentally dynamic phase after ecosystem formation and stabilized after deep-water conditions established in Lake Ohrid. As the lake deepens, we also see a switch in the macroevolutionary trade-off, resulting in a transition from a volatile assemblage of short-lived endemic species to a stable community of long-lived species. Our results emphasize the importance of the interplay between environmental/climate change, ecosystem stability, and environmental limits to diversity for diversification processes. The study also provides a new understanding of evolutionary dynamics in long-lived ecosystems.