Loricrin is the major protein of the cornified cell envelope of terminally differentiated epidermal keratinocytes which functions as a physical barrier. In order to understand its properties and role in cornified cell envelope, we have expressed human loricrin from a fulllength cDNA clone in bacteria and purified it to homogeneity. We have also isolated loricrin from newborn mouse epidermis. By circular dichroism and fluorescence spectroscopy, the in vivo mouse and bacterially expressed human loricrins possess no ␣ or ␤ structure but have some organized structure in solution associated with their multiple tyrosines and can be reversibly denatured by either guanidine hydrochloride or temperature. The transglutaminase (TGase) 1, 2, and 3 enzymes expressed during epidermal differentiation utilized loricrin in vitro as a complete substrate, but the types of cross-linking were different. The TGase 3 reaction favored certain lysines and glutamines by forming mostly intrachain cross-links, whereas TGase 1 formed mostly large oligomeric complexes by interchain crosslinks involving different lysines and glutamines. Together, the glutamines and lysines used in vitro are almost identical to those seen in vivo. The data support a hypothesis for the essential and complementary roles of both TGase 1 and TGase 3 in cross-linking of loricrin in vivo. Failure to cross-link loricrin by TGase 1 may explain the phenotype of lamellar ichthyosis, a disease caused by mutations in the TGase 1 gene.Terminal differentiation in the epidermis involves the expression of a number of specific proteins that ultimately fulfill different structural roles in the cornified, dead stratum corneum cell. One set of proteins is the keratin intermediate filaments and the interfilamentous matrix protein filaggrin (1-3). A second set of proteins is used to construct the cornified cell envelope (CE), 1 a 15-nm-thick layer of protein deposited on the inner surface of the cell periphery, which serves as a physical barrier for the epidermis (4, 5). The CE proteins are rendered insoluble by cross-linking by both disulfide bonds and the N ⑀ -(␥-glutamyl)lysine isopeptide bond formed by the action of one or more of the three known epidermal transglutaminases (TGases) (4 -6). Several proteins have now been documented as CE constituents by direct sequencing analyses of cross-linked peptides (7), including loricrin, small proline-rich proteins 1 and 2 (SPR1 and SPR2), elafin, keratins, filaggrin, and desmoplakin. The proteins involucrin and cystatin ␣ are also likely constituents, but direct sequencing of cross-linked peptides involving these proteins has not yet been reported (reviewed in Ref. 8).In particular, a variety of data have suggested that loricrin comprises about 75% of the total CE protein mass (reviewed in Ref. 9), or 85-95% of the cytoplasmic two-thirds of the CE. In fact, amino acid sequencing of many peptides recovered by the proteolysis has now provided rigorous support for this idea (7). About 90% of the molar mass of peptides from the cytoplasmic...

Peptidylarginine deiminases, which are commonly found in mammalian cells, catalyze the deimination of protein-bound arginine residues to citrullines. However, very little is known about their substrate requirements and the significance or consequences of this postsynthetic modification. We have explored this reaction in vitro with two known substrates filaggrin and trichohyalin. First, the degree and rate of modification of arginines to citrullines directly correlates with the structural order of the substrate. In filaggrin, which has little structural order, the reaction proceeded rapidly to >95% completion. However, in the highly ␣-helical protein trichohyalin, the reaction proceeded slowly to about 25% and could be forced to a maximum of about 65%. Second, the rate and degree of modification depends on the sequence location of the target arginines. Third, we show by gel electrophoresis, circular dichroism, and fluorescence spectroscopy that the reaction interferes with organized protein structure: the net formation of 10% citrulline results in protein denaturation. Cyanate modification of the lysines in model ␣-helix-rich proteins to homocitrullines also results in loss of organized structure. These data suggest that the ureido group on the citrulline formed by the peptidylarginine deiminase enzyme modification functions to unfold proteins due to decrease in net charge, loss of potential ionic bonds, and interference with H bonds.

Presteady-state and steady-state kinetic studies performed on human glutathione transferase P1-1 (EC 2.5.1.18) with 1-chloro-2,4-dinitrobenzene as co-substrate indicate that the rate-determining step is a physical event that occurs after binding of the two substrates and before the -complex formation. It may be a structural transition involving the ternary complex. This event can be related to diffusion-controlled motions of protein portions as k cat°/ k cat linearly increases by raising the relative viscosity of the solution. Similar viscosity dependence has been observed for K m GSH , while K m CDNB is independent. No change of the enzyme structure by viscosogen has been found by circular dichroism analysis. Thus, k cat and K m GSH seem to be related to the frequency and extent of enzyme structural motions modulated by viscosity. Interestingly, the reactivity of Cys-47 which can act as a probe for the flexibility of helix 2 is also modulated by viscosity. Its viscosity dependence parallels that observed for k cat and K m GSH , thereby suggesting a possible correlation between k cat , K m GSH, and diffusioncontrolled motion of helix 2. The viscosity effect on the kinetic parameters of C47S and C47S/C101S mutants confirms the involvement of helix 2 motions in the modulation of K m GSH, whereas a similar role on k cat cannot be ascertained unequivocally. The flexibility of helix 2 modulates also the homotropic behavior of GSH in these mutants. Furthermore, fluorescence experiments support a structural motion of about 4 Å occurring between helix 2 and helix 4 when GSH binds to the G-site.Human placental glutathione transferase P1-1 (GST) 1 (EC 2.5.1.18) is a dimeric enzyme composed of two identical subunits each containing one binding site for GSH (G-site) and a second binding site for the electrophilic co-substrate (H-site). Inspection of the three-dimensional structure indicates the presence near the G-site of the irregular ␣ helix 2 (residues 37-46) which is exposed to the solvent (1). Lys-44, a part of this helix, is involved in the binding of GSH. At the end of helix 2 is Cys-47 which is probably linked by ion pair formation with Lys-54. This electrostatic interaction seems important for the correct spatial arrangement of the G-site (2); lack of this bond by replacement of Cys-47 or Lys-54 with Ser or Ala lowers the affinity for GSH and triggers a positive cooperativity toward the binding of GSH (3, 4). We therefore suggested that Cys-47 acts as a hinge which limits the extent or frequency of conformational transitions involving helix 2 (3, 4). In its absence, helix 2 would become more flexible and contact the adjacent subunit via helix 4 thereby inducing the observed cooperativity (4). Several pieces of evidence indicate that the irregular ␣ helix 2 is a flexible region even in the wild-type enzyme; it displays the highest temperature factors among all other regions of domain I (5); moreover, it offers the sole point of attack (Lys-44) for the proteolytic cleavage by trypsin (5). Finally, the strongest evide...

Why are there so many dimeric proteins and enzymes? While for heterodimers a functional explanation seems quite reasonable, the case of homodimers is more puzzling. The number of homodimers found in all living organisms is rapidly increasing. A thorough inspection of the structural data from the available literature and stability (measured from denaturation–renaturation experiments) allows one to suggest that homodimers can be divided into three main types according to their mass and the presence of a (relatively) stable monomeric intermediate in the folding–unfolding pathway. Among other explanations, we propose that an essential advantage for a protein being dimeric may be the proper and rapid assembly in the cellular milieu.

Several studies have shown that anions induce collapse of acid-denatured cytochrome c into the compact A state having the properties of the molten globule and that the anion charge is the main determinant for the A state stabilization. The results here reported show that the anion size plays a role in determining the overall structure of the A state. In particular, small anions induce formation of an A state in which the native Met80-Fe(III) axial bond is recovered and the nativelike redox properties restored. On the other hand, the A state stabilized by large anions shows a histidine (His26 or His33) as the sixth ligand of the heme-iron, a very weak interaction between Trp59 and the heme propionate, and lacks nativelike redox properties. The two anion-stabilized states show similar stability, indicating that (i) the hydrophobic core (which is equally stabilized by all the anions investigated, independently of their size) is the region that mainly contributes to the macromolecule stabilization, and (ii) the flexible loops are responsible for the spectroscopic (and, thus, structural) and redox differences observed.