Trimethylamine N-oxide (TMAO) doubly locks the hydrophobic core and surfaces of protein against desiccation stress.

Interactions between proteins and osmolytes are ubiquitous within cells, assisting in response to environmental stresses. However, our understanding of protein-osmolyte interactions underlying desiccation tolerance is limited. Here, we employ solid-state NMR (ssNMR) to derive information about protein conformation and site-specific interactions between the model protein, SH3, and the osmolyte trimethylamine N-oxide (TMAO).

Quantitative entropy-enthalpy compensation in intraprotein interactions from model compound data.

Many small globular proteins exist in only two states-the physiologically relevant folded state and an inactive unfolded state. The active state is stabilized by numerous weak attractive contacts, including hydrogen bonds, other polar interactions, and the hydrophobic effect. Knowledge of these interactions is key to understanding the fundamental equilibrium thermodynamics of protein folding and stability.

Protecting Lyophilized Adenylate Kinase.

Drying protein-based drugs, usually via lyophilization, can facilitate storage at ambient temperature and improve accessibility but many proteins cannot withstand drying and must be formulated with protective additives called excipients. However, mechanisms of protection are poorly understood, precluding rational formulation design. To better understand dry proteins and their protection, we examine adenylate kinase (AdK) lyophilized alone and with the additives trehalose, maltose, bovine serum albumin, cytosolic abundant heat soluble protein D, histidine, and arginine.

Protecting Proteins from Desiccation Stress Using Molecular Glasses and Gels.

Faced with desiccation stress, many organisms deploy strategies to maintain the integrity of their cellular components. Amorphous glassy media composed of small molecular solutes or protein gels present general strategies for protecting against drying. We review these strategies and the proposed molecular mechanisms to explain protein protection in a vitreous matrix under conditions of low hydration.

H, C, N backbone resonance assignment of Escherichia coli adenylate kinase.

Adenylate kinase reversibly catalyzes the conversion of ATP plus AMP to two ADPs. This essential catalyst is present in every cell, and the Escherichia coli protein is often employed as a model enzyme. Our aim is to use the E.

Trifluoroethanol and the behavior of a tardigrade desiccation-tolerance protein.

The cosolvent 2,2,2-trifluoroethanol (TFE) is often used to mimic protein desiccation. We assessed the effects of TFE on cytosolic abundant heat soluble protein D (CAHS D) from tardigrades. CAHS D is a member of a unique protein class that is necessary and sufficient for tardigrades to survive desiccation.

Properties of a tardigrade desiccation-tolerance protein aerogel.

Lyophilization is promising for tackling degradation during the drying and storage of protein-based drugs. Tardigrade cytosolically abundant heat soluble (CAHS) proteins are necessary and sufficient for desiccation-tolerance in vivo and protein protection in vitro. Hydrated CAHS proteins form coiled-coil-based fine-stranded, cold-setting hydrogels, but the dried protein remains largely uncharacterized.

Macromolecular Crowding by Polyethylene Glycol Reduces Protein Breathing.

Most efforts to understand macromolecular crowding focus on global (i.e., complete) unfolding, but smaller excursions, often called breathing, promote aggregation, which is associated with several diseases and the bane of pharmaceutical and commercial protein production.

How Sugars Protect Dry Protein Structure.

Extremotolerant organisms and industry exploit sugars as desiccation protectants, with trehalose being widely used by both. How sugars, in general, and the hydrolytically stable sugar trehalose, in particular, protect proteins is poorly understood, which hinders the rational design of new excipients and implementation of novel formulations for preserving lifesaving protein drugs and industrial enzymes. We employed liquid-observed vapor exchange nuclear magnetic resonance (LOVE NMR), differential scanning calorimetry (DSC), and thermal gravimetric analysis (TGA) to show how trehalose and other sugars protect two model proteins: the B1 domain of streptococcal protein G (GB1) and truncated barley chymotrypsin inhibitor 2 (CI2).

Disordered proteins mitigate the temperature dependence of site-specific binding free energies.

Biophysical characterization of protein-protein interactions involving disordered proteins is challenging. A common simplification is to measure the thermodynamics and kinetics of disordered site binding using peptides containing only the minimum residues necessary. We should not assume, however, that these few residues tell the whole story.

Resolving the enthalpy of protein stabilization by macromolecular crowding.

Proteins in the cellular milieu reside in environments crowded by macromolecules and other solutes. Although crowding can significantly impact the protein folded state stability, most experiments are conducted in dilute buffered solutions. To resolve the effect of crowding on protein stability, we use F nuclear magnetic resonance spectroscopy to follow the reversible, two-state unfolding thermodynamics of the N-terminal Src homology 3 domain of the Drosophila signal transduction protein drk in the presence of polyethylene glycols (PEGs) of various molecular weights and concentrations.

Secondary structure and stability of a gel-forming tardigrade desiccation-tolerance protein.

Protein-based pharmaceuticals are increasingly important, but their inherent instability necessitates a "cold chain" requiring costly refrigeration during production, shipment, and storage. Drying can overcome this problem, but most proteins need the addition of stabilizers, and some cannot be successfully formulated. Thus, there is a need for new, more effective protective molecules.

C-terminal truncation modulates α-Synuclein's cytotoxicity and aggregation by promoting the interactions with membrane and chaperone.

α-Synuclein (α-syn) is the main protein component of Lewy bodies, the major pathological hallmarks of Parkinson's disease (PD). C-terminally truncated α-syn is found in the brain of PD patients, reduces cell viability and tends to form fibrils. Nevertheless, little is known about the mechanisms underlying the role of C-terminal truncation on the cytotoxicity and aggregation of α-syn.

Desiccation-tolerance and globular proteins adsorb similar amounts of water.

When exposed to desiccation stress, extremotolerant organisms from all domains of life produce protective disordered proteins with the potential to inform the design of excipients for formulating biologics and industrial enzymes. However, the mechanism(s) of desiccation protection remain largely unknown. To investigate the role of water sorption in desiccation protection, we use thermogravimetric analysis to study water adsorption by two desiccation-tolerance proteins, cytosolic abundant heat soluble protein D from tardigrades and late embryogenesis abundant protein 4 from the anhydrobiotic midge Polypedilum vanderplanki, and, as a control, the globular B1 domain of staphylococcal protein G.

Macromolecular Crowding Is More than Hard-Core Repulsions.

Cells are crowded, but proteins are almost always studied in dilute aqueous buffer. We review the experimental evidence that crowding affects the equilibrium thermodynamics of protein stability and protein association and discuss the theories employed to explain these observations. In doing so, we highlight differences between synthetic polymers and biologically relevant crowders.

A Difference between and In-Cell Protein Dimer Formation.

The high concentration of macromolecules in cells affects the stability of proteins and protein complexes via hard repulsions and chemical interactions, yet few studies have focused on chemical interactions. We characterized the domain-swapped dimer of the B1 domain of protein G in buffer and cells by using heteronuclear, multidimensional nuclear magnetic resonance spectroscopy. In buffer, the monomer is a partially folded molten globule, but that species is not observed in cells.

Protection by desiccation-tolerance proteins probed at the residue level.

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.

Water's Variable Role in Protein Stability Uncovered by Liquid-Observed Vapor Exchange NMR.

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 DO vapor.

Buffers, Especially the Good Kind.

Fifty-five years ago, Norman Good and colleagues authored a paper that fundamentally advanced wet biochemistry [Good, N. E., Winget, G.

The intracellular environment affects protein-protein interactions.

Protein-protein interactions are essential for life but rarely thermodynamically quantified in living cells. In vitro efforts show that protein complex stability is modulated by high concentrations of cosolutes, including synthetic polymers, proteins, and cell lysates via a combination of hard-core repulsions and chemical interactions. We quantified the stability of a model protein complex, the A34F GB1 homodimer, in buffer, cells and oocytes.

Dynamical spectroscopy and microscopy of proteins in cells.

With a strong understanding of how proteins fold in hand, it is now possible to ask how in-cell environments modulate their folding, binding and function. Studies accessing fast (ns to s) in-cell dynamics have accelerated over the past few years through a combination of in-cell NMR spectroscopy and time-resolved fluorescence microscopies. Here, we discuss this recent work and the emerging picture of protein surfaces as not just hydrophilic coats interfacing the solvent to the protein's core and functional regions, but as critical components in cells controlling protein mobility, function and communication with post-translational modifications.

Oocytes for Eukaryotic In-Cell NMR.

Understanding how the crowded and complex cellular milieu affects protein stability and dynamics has only recently become possible by using techniques such as in-cell nuclear magnetic resonance. However, the combination of stabilizing and destabilizing interactions makes simple predictions difficult. Here we show the potential of oocytes as an in-cell nuclear magnetic resonance model that can be widely used to measure protein stability and dynamics.

Dried Protein Structure Revealed at the Residue Level by Liquid-Observed Vapor Exchange NMR.

Water is key to protein structure and stability, yet the relationship between protein-water interactions and structure is poorly understood, in part because there are few techniques that permit the study of dehydrated protein structure at high resolution. Here, we describe liquid-observed vapor exchange (LOVE) NMR, a solution NMR-based method that provides residue-level information about the structure of dehydrated proteins. Using the model protein GB1, we show that LOVE NMR measurements reflect the fraction of the dried protein population trapped in a conformation where a given residue is protected from exchange with DO vapor.