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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 alpha-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 Ca-ATPase with PLB-(24-52) and SLN in liposomes showed that both proteins regulate the Ca-ATPase in a similar manner.

On the basis of the X‐ray structure and results from structure‐‐activity relationship studies, the following GM–CSF analogue was designed and synthesized by solid‐phase methodology: hGM–CSF[13‐31]‐Gly‐Pro‐Gly‐[103–116]‐NH2. This analogue was constructed to comprise helices A and D of the native hGM–CSF, covalently linked in an antiparallel orientation by the tripeptide spacer Gly‐Pro‐Gly, which is known as a turn‐inducing sequence. The conformational analysis of the analogue by CD spectroscopy revealed an essentially random structure in water, while α‐helix formation was observed upon addition of TFE. In 40% TFE the helix content was ∼45%. By two‐dimensional NMR experiments in 1:1 water/trifluoroethanol mixture two helical sequences were identified comprising the segments corresponding to helix A and helix D. In addition to medium‐range NOESY connectivities, a long‐range cross‐peak was found involving the leucine residues at positions 13 and 35. Based on the experimentally derived data (54 NOEs), the structure was refined by restrained molecular dynamics simulations over 120 ps at various temperatures. A representative conformation derived from the computer simulation is mainly characterized by two helical segments connected by a loop region. The overall three‐dimensional structure of the analogue is comparable to the X‐ray structure of hGM–CSF in that helices A and D are oriented in an antiparallel fashion, forming a two α‐helix bundle. Nevertheless, there are small differences in the topology of the helices between the solution structure of the designed analogue and the X‐ray structure of hGM–CSF. The possible implications of these conformational features at the effects of biological activity are discussed. © 1997 European Peptide Society and John Wiley & Sons, Ltd.

We have synthesized a series of peptides corresponding to portions of the extracellular domain of human granulocyte-macrophage colony stimulating factor receptor α subunit (hGM-CSFRα). The sequences were chosen according to the homology between hGM-CSFRα and the growth hormone receptor (GHR) and correspond to the regions reported to form the binding site of the latter receptor. The peptides were examined for their binding activity to hGM-CSF by affinity chromatography on resin-immobilized hGM-CSF and by a solid phase binding assay. Four peptides endowed with hGM-CSF binding activity were identified and the postulated homology between the binding sites of hGM-CSFRα and GHR was confirmed.