Protein-based biological drugs and many industrial enzymes are unstable, making them prohibitively expensive. Some can be stabilized by formulation with excipients, but most still require low temperature storage. In search of new, more robust excipients, we turned to the tardigrade, a microscopic animal that synthesizes cytosolic abundant heat soluble (CAHS) proteins to protect its cellular components during desiccation. We find that CAHS proteins protect the test enzymes lactate dehydrogenase and lipoprotein lipase against desiccation-, freezing-, and lyophilization-induced deactivation. Our data also show that a variety of globular and disordered protein controls, with no known link to desiccation tolerance, protect our test enzymes. Protection of lactate dehydrogenase correlates, albeit imperfectly, with the charge density of the protein additive, suggesting an approach to tune protection by modifying charge. Our results support the potential use of CAHS proteins as stabilizing excipients in formulations and suggest that other proteins may have similar potential.
Extremotolerant organisms from all domains of life produce protective intrinsically disordered proteins (IDPs) in response to desiccation stress. In vitro, many of these IDPs protect enzymes from dehydration stress better than U.S. Food and Drug Administration‐approved excipients. However, as with most excipients, their protective mechanism is poorly understood. Here, we apply thermogravimetric analysis, differential scanning calorimetry, and liquid‐observed vapor exchange (LOVE) NMR to study the protection of two model globular proteins (the B1 domain of staphylococcal protein G [GB1] and chymotrypsin inhibitor 2 [CI2]) by two desiccation‐tolerance proteins (CAHS D from tardigrades and PvLEA4 from an anhydrobiotic midge), as well as by disordered and globular protein controls. We find that all protein samples retain similar amounts of water and possess similar glass transition temperatures, suggesting that neither enhanced water retention nor vitrification is responsible for protection. LOVE NMR reveals that IDPs protect against dehydration‐induced unfolding better than the globular protein control, generally protect the same regions of GB1 and CI2, and protect GB1 better than CI2. These observations suggest that electrostatic interactions, charge patterning, and expanded conformations are key to protection. Further application of LOVE NMR to additional client proteins and protectants will deepen our understanding of dehydration protection, enabling the streamlined production of dehydrated proteins for expanded use in the medical, biotechnology, and chemical industries.
Water is essential to protein structure and stability, yet our understanding of how water shapes proteins is far from thorough. Our incomplete knowledge of protein−water interactions is due in part to a long-standing technological inability to assess experimentally how water removal impacts local protein structure. It is now possible to obtain residue-level information on dehydrated protein structures via liquid-observed vapor exchange (LOVE) NMR, a solution NMR technique that quantifies the extent of hydrogen−deuterium exchange between unprotected amide protons of a dehydrated protein and D 2 O vapor. Here, we apply LOVE NMR, Fourier transform infrared spectroscopy, and solution hydrogen−deuterium exchange to globular proteins GB1, CI2, and two variants thereof to link mutation-induced changes in the dehydrated protein structure to changes in solution structure and stability. We find that a mutation that destabilizes GB1 in solution does not affect its dehydrated structure, whereas a mutation that stabilizes CI2 in solution makes several regions of the protein more susceptible to dehydration-induced unfolding, suggesting that water is primarily responsible for the destabilization of the GB1 variant but plays a stabilizing role in the CI2 variant. Our results indicate that changes in dehydrated protein structure cannot be predicted from changes in solution stability alone and demonstrate the ability of LOVE NMR to uncover the variable role of water in protein stability. Further application of LOVE NMR to other proteins and their variants will improve the ability to predict and modulate protein structure and stability in both the hydrated and dehydrated states for applications in medicine and biotechnology.
The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. We developed and validated a novel method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and used solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. LBD Asyn fibrils comprise two protofilaments with pseudo-21 helical screw symmetry, very low twist and an interface formed by antiparallel beta strands of residues 85-93. The fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural landscape of LBD Asyn fibrils and inform further studies of disease mechanisms, imaging agents and therapeutics targeting Asyn.
TRPV5 and TRPV6 are inwardly rectifying calcium selective channels, considered as gatekeepers of epithelial calcium transport and key elements for calcium homeostasis. Intracellular calcium exert a negative control over the activity of these channels. In mammals, TRPV6 channels show a characteristic fast calcium-dependent inactivation phase, that is absent in TRPV5 channels at physiological conditions. It has been evidenced that the intracellular loop located between the transmembrane segments (TM) S2-S3 and residues downstream the TM S6 are involved in the mechanism of fast inactivation, however the molecular mechanism driving fast inactivation is not known. In the present study we establish a structural-functional correlation of this process. By means of electrophysiological recordings we identified a set of conserved residues at the Helix-Loop-Helix (HLH) domain that modulates the inactivation phenotype. Molecular dynamics simulations suggest that the inactivation phenotype can be explained by specific conformational changes induced by calcium coordination to a structural triad formed by the HLH domain, the intracellular connector between TM S2-S3, and the TRP helix. The TRPV1 is a nonselective cation channel that responds to various signals including capsaicin, heat, and low pH condition. The Cryo-EM structures of TRPV1 show twisting of transmembrane helices around the pore axis. To understand the intramolecular dynamics of the TRPV1 associating with the gating event, we adopted the Diffracted X-ray Tracking (DXT) technique. In DXT, an individual protein was labelled with gold nanocrystals. Trajectories of Laue diffraction spots from nanocrystals attached to the immobilized target proteins were investigated as the intramolecular movement of the target proteins in real time. DXT can monitor the rotational motion of the nanocrystal at several milliradian scales with two rotational axis views, tilting (q) and twisting (c) motions. The motion of TRPV1 was evaluated by mean square angular displacement (MSD) curves of the Laue diffraction spots from DXT. The slopes of the MSD curves obtained from TRPV1 were shifted upward in response to capsaicin, which reflects DXT successfully extracted the internal motion of TRPV1. We then applied a velocity dependent filtering method to sampling a biased motion. The filtering method clearly distinguished the agonist-and competitive antagonist (AMG9810) -induced intramolecular motion. The motions under the capsaicin and antagonist were À1 mrad (clockwise from the Au(111) direction) and 0.5 mrad (counterclockwise) after 3 ms, respectively. Such motions were also observed another filtered data. This raises the possibility that there are multiple modes of conformational change in TRPV1. The movement of ligand-evoked conformational changes was slower than the thermal fluctuations. The capsaicin receptor TRPV1 has been intensively studied by cryo-electron microscopy and functional tests. However, though the apo and capsaicinbound structural models are available, the dynamic process o...
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