The adaptor protein Hop mediates the association of the molecular chaperones Hsp70 and Hsp90. The TPR1 domain of Hop specifically recognizes the C-terminal heptapeptide of Hsp70 while the TPR2A domain binds the C-terminal pentapeptide of Hsp90. Both sequences end with the motif EEVD. The crystal structures of the TPR-peptide complexes show the peptides in an extended conformation, spanning a groove in the TPR domains. Peptide binding is mediated by electrostatic interactions with the EEVD motif, with the C-terminal aspartate acting as a two-carboxylate anchor, and by hydrophobic interactions with residues upstream of EEVD. The hydrophobic contacts with the peptide are critical for specificity. These results explain how TPR domains participate in the ordered assembly of Hsp70-Hsp90 multichaperone complexes.

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The human 31-amino acid integral membrane protein sarcolipin (SLN), which regulates the sarcoplasmic reticulum Ca-ATPase in fast-twitch skeletal muscle, was chemically synthesized. Appropriate synthesis and purification strategies were used to achieve high purity and satisfactory yields of this hydrophobic and poorly soluble protein.Structural and functional properties of SLN were analyzed and compared with the homologous region of human phospholamban (PLB) comprising residues Ala 24 -Leu 52 (PLB-(24-52)), the regulatory protein of the cardiac sarcoplasmic reticulum Ca-ATPase. Circular dichroism spectroscopy showed that SLN is a predominantly ␣-helical protein and that the secondary structure is highly resistant to SDS and thermal denaturation. In this respect SLN is remarkably similar to PLB-(24-52). However, SLN is monomeric in SDS gels, whereas PLB-(24 -52) shows a monomer-pentamer equilibrium typical for native PLB. Analytical ultracentrifugation experiments revealed that SLN oligomerizes in the presence of the nonionic detergents octylpolyoxyethylene and octyl glucoside in a concentration-dependent manner. No plateau was observed, and a pentameric state was only reached at much higher protein concentrations compared with PLB-(24-52). Chemical cross-linking showed that also in liposomes SLN has the ability to self-associate to oligomers. PLB-(24-52) specifically oligomerized to pentamers in the presence of octylpolyoxyethylene as well as in liposomes at low protein concentrations. In the presence of octylpolyoxyethylene pentamers were the main oligomeric species, whereas in liposomes monomers and dimers were predominant. Increasing the protein concentration led to self-association of PLB-(24-52) pentamers in the presence of octylpolyoxyethylene. Functional reconstitution of CaATPase with PLB-(24-52) and SLN in liposomes showed that both proteins regulate the Ca-ATPase in a similar manner. Phospholamban (PLB)1 and sarcolipin (SLN) are integral membrane proteins involved in the regulation of the sarcoplasmic reticulum Ca-ATPase. The transmembrane domain of the two proteins is highly conserved, but SLN lacks an extended cytoplasmic domain. Structural similarities between the sln and the plb gene, as well as the homology between the protein sequences of their products, led to the proposal that the two genes are members of a family (1). The 52-residue protein PLB is located in the sarcoplasmic reticulum of cardiac and slow-twitch skeletal muscle (2, 3). PLB is a critical regulator of the cardiac sarcoplasmic reticulum Ca-ATPase (SERCA2a) and myocardial contractility (4). Recently it has been shown for a mouse model of dilated cardiomyopathy that PLB is a key element in the progression of the disease (5). PLB binds to and inhibits SERCA2a in its unphosphorylated form, and phosphorylation of PLB removes the inhibitory effect. PLB consists of a hydrophilic N-terminal domain, located in the cytosol, and a hydrophobic C-terminal domain traversing the sarcoplasmic reticulum membrane. The latter has an ␣-helical conform...

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The sequence of apamin, an 18 residue bee venom toxin, encloses all the information required for the correct disulfidecoupled folding into the cystine-stabilized a-helical motif. Three apamin analogs, each containing a pair of selenocysteine residues replacing the related cysteines, were synthesized to mimic the three possible apamin isomers with two crossed, parallel, or consecutive disulfides, respectively. Refolding experiments clearly revealed that the redox potential of selenocysteine prevails over the sequence encoded structural information for proper folding of apamin. Thus, selenocysteine can be used as a new device to generate productive and nonproductive folding intermediates of peptides and proteins. In fact, disulfides are selectively reduced in presence of the diselenide and the conformational features derived from these intermediates as well as from the three-dimensional~3D! structures of the selenocysteine-containing analogs with their nonnatural networks of diselenide0disulfide bridges allowed to gain further insight into the subtle driving forces for the correct folding of apamin that mainly derive from local conformational preferences.