The photocycle of the photophobic receptor sensory rhodopsin II from N. pharaonis was analyzed by varying measuring wavelengths, temperature, and pH, and by exchanging H2O with D2O. The data can be satisfactorily modeled by eight exponents over the whole range of modified parameters. The kinetic data support a model similar to that of bacteriorhodopsin (BR) if a scheme of irreversible first-order reactions is assumed. Eight kinetically distinct protein states can then be identified. These states are formed from five spectrally distinct species. The chromophore states Si correspond in their spectral properties to those of the BR photocycle, namely pSRII510 (K), pSRII495 (L), pSRII400 (M), pSRII485 (N), and pSRII535 (O). In comparison to BR, pSRII400 is formed approximately 10 times faster than the M state; however, the back-reaction is almost 100 times slower. Comparison of the temperature dependence of the rate constants with those from the BR photocycle suggests that the differences are caused by changes of DeltaS. The rate constants of the pSRII photocycle are almost insensitive to the pH variation from 9.0 to 5.5, and show only a small H2O/D2O effect. This analysis supports the idea that the conformational dynamics of pSRII controls the kinetics of the photocycle of pSRII.

Protein aggregation via polyglutamine stretches occurs in a number of severe neurodegenerative diseases such as Huntington's disease. We have investigated fibrillar aggregates of polyglutamine peptides below, at, and above the toxicity limit of around 37 glutamine residues using solid-state NMR and electron microscopy. Experimental data are consistent with a dry fibril core of at least 70-80 Å in width for all constructs. Solid-state NMR dipolar correlation experiments reveal a largely β-strand character of all samples and point to tight interdigitation of hydrogen-bonded glutamine side chains from different sheets. Two approximately equally frequent populations of glutamine residues with distinct sets of chemical shifts are found, consistent with local backbone dihedral angles compensating for β-strand twist or with two distinct sets of side-chain conformations. Peptides comprising 15 glutamine residues are present as single extended β-strands. Data obtained for longer constructs are most compatible with a superpleated arrangement with individual molecules contributing β-strands to more than one sheet and an antiparallel assembly of strands within β-sheets.

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ClpB fromClpB from Thermus thermophilus is a member of the AAA protein superfamily that is important for a variety of biological activities (1). Despite their different cellular functions AAA proteins (ATPases associated with a variety of cellular activities) employ a general mechanism. They mediate the assembly and disassembly of large protein complexes that are involved in processes like DNA replication, vesicle transport, or organelle biogenesis. The AAA protein superfamily comprises the Clp/ Hsp100 proteins with its members ClpA, ClpX, and ClpY on the one hand and ClpB on the other.ClpA, ClpX, and ClpY (HslU) interact with cellular peptidases to form ATP-dependent proteases. The role of these Clp proteins is the unfolding of substrates and the delivery of unfolded polypeptides to the protease subunit. In contrast ClpB from Escherichia coli and T. thermophilus and the Saccharomyces cerevisiae homologue Hsp104 do not appear to bind to cellular proteases. Instead they interact with the DnaK/ Hsp70 chaperone system to assist the disaggregation of protein aggregates (2-6).Clp proteins are further classified according to the number of nucleotide binding domains (NBD), 1 which are 220 -250 amino acids in length and show a high level of sequence homology. Class two proteins contain only one NBD whereas the class one members ClpA and ClpB contain two NBDs. In ClpB and Hsp104 ATP hydrolysis at both NBDs was shown to be necessary for the chaperone activity of the Clp proteins (7,8).Structural information on the class two protein HslU (ClpY) and the hexamerization domain (D2) of N-ethylmaleimide-sensitive fusion protein give insight into the domain architecture of AAA proteins (9 -11). These crystal structures show ringshaped oligomers composed of six monomers. The AAA modules of both proteins show a similar overall structure, consisting of the core NBD and a C-terminal, mostly helical, domain. The core NBD contains the Rossman fold including the phosphate binding loop of ATP-and GTP-binding proteins, which consists of five central ␤-sheets flanked by ␣-helices. In the case of HslU an additional helical domain is inserted in the NBD that is not present in other Clp proteins.In ClpB Eco the C-terminal domain of the second AAA cassette was shown to be important for oligomerization, ATPase activity, and chaperone function (12). Isolated C-domains of ClpA, ClpX, and ClpY, which associate with peptidases, were found to interact with different substrate proteins in vivo. Therefore the C-terminal domains were proposed to be sensor and substrate discrimination domains although the C-terminal domains show only little sequence homology between different Clp proteins (13).The class one Clp proteins have, in addition, an N-terminal domain that precedes the first NBD. The ClpA and ClpB mRNAs of E. coli contain internal translation initiation sites and are expressed in vivo as two gene products: the full-length proteins and shortened versions lacking the N-terminal domain (14,15). The N-terminal domains are supposed to consist of two...

The blue-light receptor genes (sopII) of sensory rhodopsin (SR) II were cloned from two species, the halophilic bacteria Haloarcula vallismortis (vSR-II) and Natronobacterium pharaonis (pSR-II). Upstream of both soplI gene loci, sequences corresponding to the halobacterial transducer of rhodopsin (Htr) II In bacterial taxis, the cell recognizes and reacts actively to numerous chemical and physical stimuli. A well-studied example is found in the chemotactic behavior ofEscherichia coli. The chemoreceptors of E. coli display two functional properties, the reception of the signal on the external side of the cell and the signal transduction to cytoplasmic proteins (reviewed in ref. 1). Characteristic of transducer proteins is their ability to adapt to a constant signal input by reversible methylation of specific sites flanking a cytoplasmic domain (signal domain), which links the incoming signal with cytoplasmic components.In a similar signal transduction system, the phototactic activity of the archaeon Halobacterium salinarium is mediated by photoreceptors (reviewed in refs. 2 and 3) that have a close structural relationship to the ion pumps of bacteriorhodopsin (BR) and halorhodopsin (reviewed in ref. 4). The bacteria are attracted to light >520 nm and repelled by light <500 nm. The photoattractant response of Halobacteria is mediated by sensory rhodopsin (SR) I. In a two-photon reaction, it can also trigger a negative response toward near-UV light. Moreover, repellent light of wavelengths <500 nm is recognized by a second pigment, SR-II, which absorbs at -490 nm.

Conversion of cellular prion protein (PrP(C)) into the pathological conformer (PrP(Sc)) has been studied extensively by using recombinantly expressed PrP (rPrP). However, due to inherent difficulties of expressing and purifying posttranslationally modified rPrP variants, only a limited amount of data is available for membrane-associated PrP and its behavior in vitro and in vivo. Here, we present an alternative route to access lipidated mouse rPrP (rPrP(Palm)) via two semisynthetic strategies. These rPrP variants studied by a variety of in vitro methods exhibited a high affinity for liposomes and a lower tendency for aggregation than rPrP. In vivo studies demonstrated that double-lipidated rPrP is efficiently taken up into the membranes of mouse neuronal and human epithelial kidney cells. These latter results enable experiments on the cellular level to elucidate the mechanism and site of PrP-PrP(Sc) conversion.

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