The S100 protein family consists of 24 members functionally distributed into three main subgroups: those that only exert intracellular regulatory effects, those with intracellular and extracellular functions and those which mainly exert extracellular regulatory effects. S100 proteins are only expressed in vertebrates and show cell-specific expression patterns. In some instances, a particular S100 protein can be induced in pathological circumstances in a cell type that does not express it in normal physiological conditions. Within cells, S100 proteins are involved in aspects of regulation of proliferation, differentiation, apoptosis, Ca2+ homeostasis, energy metabolism, inflammation and migration/invasion through interactions with a variety of target proteins including enzymes, cytoskeletal subunits, receptors, transcription factors and nucleic acids. Some S100 proteins are secreted or released and regulate cell functions in an autocrine and paracrine manner via activation of surface receptors (e.g. the receptor for advanced glycation end-products and toll-like receptor 4), G-protein-coupled receptors, scavenger receptors, or heparan sulfate proteoglycans and N-glycans. Extracellular S100A4 and S100B also interact with epidermal growth factor and basic fibroblast growth factor, respectively, thereby enhancing the activity of the corresponding receptors. Thus, extracellular S100 proteins exert regulatory activities on monocytes/macrophages/microglia, neutrophils, lymphocytes, mast cells, articular chondrocytes, endothelial and vascular smooth muscle cells, neurons, astrocytes, Schwann cells, epithelial cells, myoblasts and cardiomyocytes, thereby participating in innate and adaptive immune responses, cell migration and chemotaxis, tissue development and repair, and leukocyte and tumor cell invasion.

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.

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...