@article {145428, title = {Factors affecting macromolecule orientations in thin films formed in cryo-EM.}, journal = {Acta Crystallogr D Struct Biol}, volume = {80}, year = {2024}, month = {2024 Jul 01}, pages = {535-550}, abstract = {

The formation of a vitrified thin film embedded with randomly oriented macromolecules is an essential prerequisite for cryogenic sample electron microscopy. Most commonly, this is achieved using the plunge-freeze method first described nearly 40 years ago. Although this is a robust method, the behaviour of different macromolecules shows great variation upon freezing and often needs to be optimized to obtain an isotropic, high-resolution reconstruction. For a macromolecule in such a film, the probability of encountering the air-water interface in the time between blotting and freezing and adopting preferred orientations is very high. 3D reconstruction using preferentially oriented particles often leads to anisotropic and uninterpretable maps. Currently, there are no general solutions to this prevalent issue, but several approaches largely focusing on sample preparation with the use of additives and novel grid modifications have been attempted. In this study, the effect of physical and chemical factors on the orientations of macromolecules was investigated through an analysis of selected well studied macromolecules, and important parameters that determine the behaviour of proteins on cryo-EM grids were revealed. These insights highlight the nature of the interactions that cause preferred orientations and can be utilized to systematically address orientation bias for any given macromolecule and to provide a framework to design small-molecule additives to enhance sample stability and behaviour.

}, keywords = {Animals, Cryoelectron Microscopy, Freezing, Macromolecular Substances, Proteins, Vitrification}, issn = {2059-7983}, doi = {10.1107/S2059798324005229}, author = {Yadav, Swati and Vinothkumar, Kutti R} } @article {95518, title = {Biophysical Properties of the Isolated Spike Protein Binding Helix of Human ACE2.}, journal = {Biophys J}, year = {2021}, month = {2021 Jun 29}, abstract = {

The entry of the SARS-CoV2 virus in human cells is mediated by the binding of its surface spike protein to the human Angiotensin-Converting Enzyme 2 (ACE2) receptor. A 23 residues long helical segment (SBP1) at the binding interface of human ACE2 interacts with viral spike protein and therefore, has generated considerable interest as a recognition element for virus detection. Unfortunately, emerging reports indicate that the affinity of SBP1 to the receptor-binding domain (RBD) of the spike protein is much lower than that of the ACE2 receptor itself. Here, we examine the biophysical properties of SBP1 to reveal factors leading to its low affinity for the spike protein. While SBP1 shows good solubility (solubility \> 0.8 mM), CD spectroscopy shows that it is mostly disordered with some anti-parallel beta-sheet content, and no helicity. The helicity is substantial (\> 20\%) only upon adding high concentrations (>= 20\% v/v) of 2,2,2-trifluoroethanol, a helix-promoter. Fluorescence correlation spectroscopy and single molecule photobleaching studies show that the peptide oligomerizes at concentrations \> 50 nM. We hypothesized that mutating the hydrophobic residues (F28, F32, and F40) of SBP1 which do not directly interact with the spike protein to alanine would reduce peptide oligomerization without affecting its spike binding affinity. While the mutant peptide (SBP1) shows substantially reduced oligomerization propensity, it does not show improved helicity. Our study shows that the failure of efforts so far to produce a short SBP1 mimic with a high affinity for the spike protein is not only due to the lack of helicity, but also due to the heretofore unrecognized problem of oligomerization.

}, issn = {1542-0086}, doi = {10.1016/j.bpj.2021.06.017}, author = {Das, Anirban and Vishvakarma, Vicky and Dey, Arpan and Dey, Simli and Gupta, Ankur and Das, Mitradip and Vishwakarma, Krishna Kant and Roy, Debsankar Saha and Yadav, Swati and Kesarwani, Shubham and Venkatramani, Ravindra and Maiti, Sudipta} } @article {79721, title = {A 2-Tyr-1-carboxylate Mononuclear Iron Center Forms the Active Site of a Paracoccus Dimethylformamidase.}, journal = {Angew Chem Int Ed Engl}, year = {2020}, month = {2020 May 25}, abstract = {

N,N-dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved to use DMF as a sole carbon and nitrogen source for growth via degradation by a dimethylformamidase (DMFase). We show that DMFase from Paracoccus sp. strain DMF is a halophilic and thermostable enzyme comprising a multimeric complex of the α β or (α β ) type. One of the three domains of the large subunit and the small subunit are hitherto undescribed protein folds of unknown evolutionary origin. The active site consists of a mononuclear iron coordinated by two Tyr side-chain phenolates and one carboxylate from Glu. The Fe ion in the active site catalyzes the hydrolytic cleavage of the amide bond in DMF. Kinetic characterization reveals that the enzyme shows cooperativity between subunits, and mutagenesis and structural data provide clues to the catalytic mechanism.

}, issn = {1521-3773}, doi = {10.1002/anie.202005332}, author = {Arya, Chetan Kumar and Yadav, Swati and Fine, Jonathan and Casanal, Ana and Chopra, Gaurav and Ramanathan, Gurunath and Vinothkumar, Kutti R and Subramanian, Ramaswamy} }