Internal ionizable groups in proteins are relatively rare but they are essential for catalysis and energy transduction. To examine molecular determinants of their unusual and functionally important properties, we engineered 25 variants of staphylococcal nuclease with lysine residues at internal positions. Nineteen of the Lys residues have depressed pK a values, some as low as 5.3, and 20 titrate without triggering any detectable conformational reorganization. Apparently, simply by being buried in the protein interior, these Lys residues acquired pK a values comparable to those of naturally occurring internal ionizable groups involved in catalysis and biological H þ transport. The pK a values of some of the internal Lys residues were affected by interactions with surface carboxylic groups. The apparent polarizability reported by the pK a values varied significantly from location to location inside the protein. These data will enable an unprecedented examination of the positional dependence of the dielectric response of a protein. This study also shows that the ability of proteins to withstand the presence of charges in their hydrophobic interior is a fundamental property inherent to all stable proteins, not a specialized adaptation unique to proteins that evolved to depend on internal charges for function.

Under stress, certain eukaryotic proteins and RNA assemble to form membraneless organelles known as stress granules. The most well-studied stress granule components are RNA-binding proteins that undergo liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by intrinsically disordered low-complexity domains (LCDs). Here we show that stress granules include proteasomal shuttle factor UBQLN2, an LCD-containing protein structurally and functionally distinct from RNA-binding proteins. In vitro, UBQLN2 exhibits LLPS at physiological conditions. Deletion studies correlate oligomerization with UBQLN2's ability to phase-separate and form stress-induced cytoplasmic puncta in cells. Using nuclear magnetic resonance (NMR) spectroscopy, we mapped weak, multivalent interactions that promote UBQLN2 oligomerization and LLPS. Ubiquitin or polyubiquitin binding, obligatory for UBQLN2's biological functions, eliminates UBQLN2 LLPS, thus serving as a switch between droplet and disperse phases. We postulate that UBQLN2 LLPS enables its recruitment to stress granules, where its interactions with ubiquitinated substrates reverse LLPS to enable shuttling of clients out of stress granules.

Charges are inherently incompatible with hydrophobic environments. Presumably for this reason, ionizable residues are usually excluded from the hydrophobic interior of proteins and are found instead at the surface, where they can interact with bulk water. Paradoxically, ionizable groups buried in the hydrophobic interior of proteins play essential roles, especially in biological energy transduction. To examine the unusual properties of internal ionizable groups we measured the pK a of glutamic acid residues at 25 internal positions in a stable form of staphylococcal nuclease. Two of 25 Glu residues titrated with normal pK a near 4.5; the other 23 titrated with elevated pK a values ranging from 5.2-9.4, with an average value of 7.7. Trp fluorescence and far-UV circular dichroism were used to monitor the effects of internal charges on conformation. These data demonstrate that although charges buried in proteins are indeed destabilizing, charged side chains can be buried readily in the hydrophobic core of stable proteins without the need for specialized structural adaptations to stabilize them, and without inducing any major conformational reorganization. The apparent dielectric effect experienced by the internal charges is considerably higher than the low dielectric constants of hydrophobic matter used to represent the protein interior in electrostatic continuum models of proteins. The high thermodynamic stability required for proteins to withstand the presence of buried charges suggests a pathway for the evolution of enzymes, and it underscores the need to mind thermodynamic stability in any strategy for engineering novel or altered enzymatic active sites in proteins.dielectric effect | electrostatics | hydration | pKa | bioenergetics T he transfer of an ion from water into a less polar and polarizable environment, such as the hydrophobic interior of a protein, is energetically unfavorable. Internal charges usually destabilize the folded states of proteins, which is primarily why charged groups are largely excluded from the hydrophobic interior and found instead at the protein-water interface, where they can interact with bulk water (1). Paradoxically, internal ionizable groups in proteins are essential for biological energy transduction. These type of ionizable groups are found in the active sites of enzymes (2), and are necessary for e − transfer and H þ transport in proteins such as ATPase (3) and cytochrome c oxidase (4), for ion homeostasis (5, 6), and for light-activated processes in proteins such as bacteriorhodopsin (7,8). The structural adaptations necessary for proteins to tolerate internal ionizable groups, and the factors that stabilize internal charges, are poorly understood. For this reason, our understanding of fundamental aspects of function and evolution of proteins is still limited, as is our ability to manipulate and design novel enzymes.To examine systematically the capacity of globular proteins to tolerate the presence of buried charges, we measured the pK a of 25 internal glutamic acid resid...

Prior computational studies of the acid-unfolding behavior of staphylococcal nuclease (SNase) suggest that the pK(a) values of its carboxylic groups are difficult to reproduce with electrostatics calculations with continuum methods. To examine the molecular determinants of the pK(a) values of carboxylic groups in SNase, the pK(a) values of all 20 Asp and Glu residues were measured with multidimensional and multinuclear NMR spectroscopy in an acid insensitive variant of SNase. The crystal structure of the protein was obtained to describe the microenvironments of the carboxylic groups. Fourteen Asp and Glu residues titrate with relatively normal pK(a) values that are depressed by less than 1.1 units relative to the normal pK(a) of Asp and Glu in water. Only six residues have pK(a) values shifted by more than 1.5 units. Asp-21 has an unusually high pK(a) of 6.5, which is probably the result of interactions with other carboxylic groups at the active site. The most perturbed pK(a) values appear to be governed by hydrogen bonding and not by Coulomb interactions. The pK(a) values calculated with standard continuum electrostatics methods applied to static structures are more depressed than the measured values because Coulomb effects are exaggerated in the calculations. The problems persist even when the protein is treated with the dielectric constant of water. This can be interpreted to imply that structural relaxation is an important determinant of the pK(a) values; however, no major pH-sensitive conformational reorganization of the backbone was detected using NMR spectroscopy.