Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and a primary cause of pneumonia and urinary tract infections in intensive care units (1, 2). Among the most vulnerable individuals are those with immunodeficiency, cystic fibrosis, or severe burns or who require mechanical ventilation. A major virulence determinant of P. aeruginosa is a type III secretion system (T3SS). 2 The T3SS encodes a multiprotein complex, called an injectisome, which functions by injecting or translocating effector proteins directly into the cytoplasm of eukaryotic host cells. The T3SS of P. aeruginosa is used to translocate four known effectors that subvert signal transduction pathways to inhibit phagocytosis and elicit cytotoxicity toward host cells (3, 4).Expression of the P. aeruginosa T3SS is highly regulated and under the direct transcriptional control of ExsA, a member of the AraC family of transcriptional activators (5). Environmental signals, including contact of P. aeruginosa with host cells and Ca 2ϩ -limiting growth conditions, induce ExsA-dependent transcription of the T3SS (6, 7). Although the mechanism of induction by host cell contact is unclear, Ca 2ϩ depletion regulates T3SS gene transcription by inducing type III secretory activity (8). Transcription of T3SS genes is intimately coupled to type III secretory activity through a regulatory cascade that governs ExsA activity. This regulatory cascade consists of three interacting proteins (ExsD, ExsC, and ExsE). ExsD functions as an anti-activator by directly binding to and inhibiting ExsA-dependent transcription (8). ExsC functions as an anti-anti-activator by binding to and antagonizing ExsD activity (9). Finally, ExsE is an inhibitor of ExsC activity and a secreted substrate of the T3SS (10, 11). Dasgupta et al. (9) have proposed the following model to account for transcriptional induction of T3SS genes by Ca 2ϩ depletion. Under Ca 2ϩ replete conditions, ExsE is retained in the cytoplasm because of the lack of type III secretory activity and forms a complex with ExsC. The sequestration of ExsC by ExsE allows for the binding of the anti-activator ExsD to ExsA, thereby blocking transcription. In response to Ca 2ϩ depletion, however, ExsE is secreted into the extracellular milieu. The corresponding decrease in intracellular ExsE levels releases ExsC, which then sequesters ExsD and thereby makes ExsA available to activate transcription of T3SS genes.A common feature of proteins secreted by the T3SS is the requirement for chaperone activity (12). ExsC has biochemical characteristics of a type III-specific chaperone and is required for ExsE stability within the cytoplasm and for ExsE secretion (9, 10). T3SS-specific chaperones fall into one of three classes based on substrate specificity. Class I, II, and III chaperones facilitate secretion of effectors, components of the transloca-

In mediating protein folding, chaperonin GroEL and cochaperonin GroES form an enclosed chamber for substrate proteins in an ATP-dependent manner. The essential role of the double ring assembly of GroEL is demonstrated by the functional deficiency of the single ring GroEL SR . The GroEL SR -GroES is highly stable with minimal ATPase activity. To restore the ATP cycle and the turnover of the folding chamber, we sought to weaken the GroEL SR -GroES interaction systematically by concatenating seven copies of groES to generate groES 7 . GroES Ile-25, Val-26, and Leu-27, residues on the GroEL-GroES interface, were substituted with Asp on different groES modules of groES 7 . GroES 7

A novel peptide, the backward reading sequence of human metallothionein-2 a domain, was synthesized and its chemical and spectroscopic properties analyzed. This folded retro-a domain was able to bind Cd(II) in identical stoichiometries with the chemically synthesized a domain of metallothionein-2. Nearly identical to the a domain, Cd-binding retro-a domain showed a characteristic ultraviolet absorption spectrum with a shoulder at 245±250 nm (due to cadmium±thiolate charge transfer), and the absorption shoulder was abolished by acidification [suggesting mercaptide bonding between Cd(II) and the cysteine residues]. Similar metal-binding capabilities between a domain and retro-a domain were observed also by pH titration and in the reaction with the sulfhydryl reagent 5,5H -dithiobis(2-nitrobenzoic acid). A two-state cooperativity of the metal-cluster formation was observed spectroscopically in the titration of the retro-a domain, indicating that the retro-protein is foldable. In contrast to other proteins, our results indicate that the reversion of the amino acid sequence for the a domain does not change its foldability and metal-binding capacity, suggesting that the order of its sequence is not critical to the formation of a critical metal±tetrathiolate nucleus. However, CD spectra of the Cd-binding a domain and retro-a domain showed that the reversal direction of the domain sequence backbone significantly affects the formation of structure even when it is foldable.Keywords: folding nucleus; metallothionein; metal-binding protein; protein design; retro-protein.A retro-protein is a peptide chain altered by a reversal of the direction of the amino acid sequence. The chirality of the side chain positions of a retro-protein is equivalent to that of a d-protein [1]. d-proteins acquire the perfect mirror image structures of their corresponding, natural l-proteins [2]. Thus, theoretically, a retro-protein might adapt a topological equivalent of the mirror image of the three-dimensional structure of its parent protein [1,3]. Lattice model simulations have predicted that the topology of a retro-protein is that of the native protein, rather than that corresponding to the mirror image of the native protein [4]. However, analyses of inverse sequence similarity in proteins show that the tertiary structures of retro-proteins did not imply folds comparable to their parent protein [5]. Recent results indicate that the sequence inversion affects the foldability of some model peptides, and these retro-proteins are no more similar to their parent sequences than any random sequence, despite their common hydrophobic/hydrophilic pattern, global amino acid composition and possible tertiary contacts. Retro sequences of the B domain of Staphylococcal protein A and the SH3 domain of a-spectrin for instance were unfolded, indicating foldability of the protein [3]. However, as only a few sequences have been examined, it remains possible that certain retro-proteins are foldable given the presence of a specific nucleus, such as a geometrically...

The objective of this study was to identify characteristics of informal caregivers, caregiving, and the people with multiple sclerosis (MS) receiving assistance that are associated with reduced caregiver employment. Data were collected during telephone interviews with 530 MS caregivers, including 215 employed caregivers, with these survey data analyzed using logistic regression. Poorer cognitive ability by the care recipient to make decisions about daily tasks and more caregiving hours per week predicted reduced caregiver employment. Better physical health domains of caregiver quality of life were associated with significantly lower odds of reduced employment. Health professionals treating informal caregivers, as well as those treating people with MS, need to be aware of respite, support, and intervention programs available to MS caregivers and refer them to these programs, which could reduce the negative impact of caregiving on employment.

Many Gram-negative bacteria utilize a type III secretion system (T3SS) to deliver protein effectors to target host cells. Transcriptional control of T3SS gene expression is generally coupled to secretion through the release of a regulatory protein. T3SS gene expression in Pseudomonas aeruginosa is regulated by extracellular secretion of ExsE. ExsE is a small 81 residue protein that appears to lack a stable structural core as indicated by previous studies. In this study, we employed various NMR methods to characterize the structure of ExsE alone and when bound to its secretion chaperone ExsC. We found that ExsE is largely unfolded throughout the polypeptide chain, belonging to a class of proteins that are intrinsically disordered. The unfolded, extended conformation of ExsE may expedite efficient secretion through the narrow path of the T3SS secretion channel to activate gene expression in a timely manner. We also found that the structurally flexible ExsE samples through conformations with localized structurally ordered regions. Importantly, these transiently ordered elements are related to the secondary structures involved in binding ExsC based on a prior crystal structure of the ExsCExsE complex. These findings support the notion that preexisting structured elements facilitate binding of intrinsically disordered proteins to their targets.