Investigation of Indazole Unbinding Pathways in CYP2E1 by Molecular Dynamics Simulations

Human microsomal cytochrome P450 2E1 (CYP2E1) can oxidize not only low molecular weight xenobiotic compounds such as ethanol, but also many endogenous fatty acids. The crystal structure of CYP2E1 in complex with indazole reveals that the active site is deeply buried into the protein center. Thus, the unbinding pathways and associated unbinding mechanisms remain elusive. In this study, random acceleration molecular dynamics simulations combined with steered molecular dynamics and potential of mean force calculations were performed to identify the possible unbinding pathways in CYP2E1. The results show that channel 2c and 2a are most likely the unbinding channels of CYP2E1. The former channel is located between helices G and I and the B-C loop, and the latter resides between the region formed by the F-G loop, the B-C loop and the β1 sheet. Phe298 and Phe478 act as the gate keeper during indazole unbinding along channel 2c and 2a, respectively. Previous site-directed mutagenesis experiments also supported these findings

Adult survival and annual movement patterns of common snipe in Iceland

The common snipe (Gallinago gallinago) is a wader that breeds in subarctic regions from Iceland to Russia, and for which global populations are in decline. We studied snipe breeding in western Iceland between 1998 and 2020, locating nests and ringing birds annually. In 2019 and 2020, we deployed geolocators on nesting adults to estimate the timing of their annual migration and the location of overwintering areas. Birds moved principally between breeding locations in Iceland to wintering areas in Ireland, although some birds may winter farther north. We also found that apparent annual adult survival averaged 66%, but was higher in years with warmer, wetter winters. Given the similarity of our survival estimates to those from snipe elsewhere, we suggest that adult survival is unlikely a major contributor to declining populations, and other factors like habitat loss may be of more concern

Pushing the limits of automatic computational protein design: design, expression, and characterization of a large synthetic protein based on a fungal laccase scaffold

The de novo engineering of new proteins will allow the design of complex systems in synthetic biology. But the design of large proteins is very challenging due to the large combinatorial sequence space to be explored and the lack of a suitable selection system to guide the evolution and optimization. One way to approach this challenge is to use computational design methods based on the current crystallographic data and on molecular mechanics. We have used a laccase protein fold as a scaffold to design a new protein sequence that would adopt a 3D conformation in solution similar to a wild-type protein, the Trametes versicolor (TvL) fungal laccase. Laccases are multi-copper oxidases that find utility in a variety of industrial applications. The laccases with highest activity and redox potential are generally secreted fungal glycoproteins. Prokaryotic laccases have been identified with some desirable features, but they often exhibit low redox potentials. The designed sequence (DLac) shares a 50% sequence identity to the original TvL protein. The new DLac gene was overexpressed in E. coli and the majority of the protein was found in inclusion bodies. Both soluble protein and refolded insoluble protein were purified, and their identity was verified by mass spectrometry. Neither protein exhibited the characteristic T1 copper absorbance, neither bound copper by atomic absorption, and neither was active using a variety of laccase substrates over a range of pH values. Circular dichroism spectroscopy studies suggest that the DLac protein adopts a molten globule structure that is similar to the denatured and refolded native fungal TvL protein, which is significantly different from the natively secreted fungal protein. Taken together, these results indicate that the computationally designed DLac expressed in E. coli is unable to utilize the same folding pathway that is used in the expression of the parent TvL protein or the prokaryotic laccases. This sequence can be used going forward to help elucidate the sequence requirements needed for prokaryotic multi-copper oxidase expression