Acid-labile protecting groups for the synthesis of lipidated peptides.

Lipidated peptides and their neolipoprotein derivatives are efficient tools for the investigation of biological processes in molecular detail. These compounds are often acid- and base-labile, and their synthesis requires the use of a combination of blocking groups that can be removed under very mild conditions. In this article we demonstrate that the Boc urethane and different trityl-type protecting groups can be cleaved selectively under acidic conditions that are mild enough to be compatible with the demands of lipopeptide synthesis. Thus, the Boc group was cleaved with TMS triflate in the presence of lutidine, and the methyltrityl (Mtt) and the methoxytrityl (Mmt) group were removed with 1% TFA in dichloromethane in the presence of triethylsilane as cation scavenger. Removal of the phenylfluorenyl group was achieved with up to 3% TFA in dichloromethane in the presence of triethylsilane at 0 degrees C. These protecting-group techniques were successfully applied in the synthesis of differently lipidated H-Ras peptides.

Linking the fields--the interplay of organic synthesis, biophysical chemistry, and cell biology in the chemical biology of protein lipidation.

Research in the biological sciences has undergone a fundamental and dramatic change during the last decades. Whereas biology was more phenomenologically oriented for a long time, today many biological processes are investigated and understood in molecular detail. It has become evident that all biological phenomena have a chemical basis: Biology is based on chemical principles. In the past, this insight had led to the development of biochemistry, molecular biology, and modern pharmacology. Today it increasingly determines the manner in which various biological phenomena are studied. The tools provided by classical biological techniques often are not sufficient to address the prevailing issues in precise molecular detail. Instead, the strengths of both chemical and biological methodology have to be used. Several recent research projects have proven that combining the power of organic synthesis with cell biology may open up entirely new and alternative opportunities for the study of biological problems. In this review we summarize the successful interplay between three disciplines-organic synthesis, biophysics, and cell biology-in the study of protein lipidation and its relevance to targeting of proteins to the plasma membrane of cells in precise molecular detail. This interplay is highlighted by using the Ras protein as a representative example. The development of methods for the synthesis of Ras-derived peptides and fully functional Ras proteins, the determination of their biophysical properties, in particular the ability to bind to model membranes, and finally the use of synthetic Ras peptides and Ras proteins in cell biological experiments are addressed. The successful combination of these three disciplines has led to a better understanding of the factors governing the selective targeting of Ras and related lipid-modified proteins to the plasma membrane.

Farnesylation of Ras is important for the interaction with phosphoinositide 3-kinase gamma.

The correct functioning of Ras proteins requires post-translational modification of the GTP hydrolases (GTPases). These modifications provide hydrophobic moieties that lead to the attachment of Ras to the inner side of the plasma membrane. In this study we investigated the role of Ras processing in the interaction with various putative Ras-effector proteins. We describe a specific, GTP-independent interaction between post-translationally modified Ha- and Ki-Ras4B and the G-protein responsive phosphoinositide 3-kinase p110gamma. Our data demonstrate that post-translational processing increases markedly the binding of Ras to p110gamma in vitro and in Sf9 cells, whereas the interaction with p110alpha is unaffected under the same conditions. Using in vitro farnesylated Ras, we show that farnesylation of Ras is sufficient to produce this effect. The complex of p110gamma and farnesylated RasGTP exhibits a reduced dissociation rate leading to the efficient shielding of the GTPase from GTPase activating protein (GAP) action. Moreover, Ras processing affects the dissociation rate of the RasGTP complex with the Ras binding domain (RBD) of Raf-1, indicating that processing induces alterations in the conformation of RasGTP. The results suggest a direct interaction between a moiety present only on fully processed or farnesylated Ras and the putative target protein p110gamma.