The recombinant HIV-1 Tat protein contains a small region corresponding to residues 47 YGRKKRRQRR 57 R, which is capable of translocating cargoes of different molecular sizes, such as proteins, DNA, RNA, or drugs, across the cell membrane in an apparently energy-independent manner. The pathway that these peptides follow for entry into the cell has been the subject of strong controversy for the last decade. This peptide is highly basic and hydrophilic. Therefore, a central question that any candidate mechanism has to answer is how this highly hydrophilic peptide is able to cross the hydrophobic barrier imposed by the cell membrane. We propose a mechanism for the spontaneous translocation of the Tat peptides across a lipid membrane. This mechanism involves strong interactions between the Tat peptides and the phosphate groups on both sides of the lipid bilayer, the insertion of charged side chains that nucleate the formation of a transient pore, followed by the translocation of the Tat peptides by diffusing on the pore surface. This mechanism explains how key ingredients, such as the cooperativity among the peptides, the large positive charge, and specifically the arginine amino acids, contribute to the uptake. The proposed mechanism also illustrates the importance of membrane fluctuations. Indeed, mechanisms that involve large fluctuations of the membrane structure, such as transient pores and the insertion of charged amino acid side chains, may be common and perhaps central to the functions of many membrane protein functions.cell-penetrating peptide ͉ antimicrobial peptide ͉ drug delivery ͉ membrane proteins ͉ pore formation T he HIV-1 Tat protein indicated that some proteins might have short sequence segments responsible for their translocation across cell membranes in a seemingly energy-independent manner (1-8). The sequence responsible for the cellular uptake of the HIV-1 Tat corresponds to residues 47 YGRKKRRQRR 57 R. This peptide is called the Tat peptide, and it has been categorized as a member of a family of peptides called cell-penetrating peptides. These peptides consist of short sequences (10-30 aa) that are highly basic and enter the cells in a seemingly energy-independent manner (1). This property makes them extraordinarily good candidates as transporters for drug delivery (7, 9-11). Therefore, significant effort is currently being invested to understand this phenomenon at a fundamental molecular level, as well as to employ these peptides as drug carriers (2,(12)(13)(14)(15)(16).Much debate exists around what makes it possible for these peptides to translocate across biological membranes. A diverse set of experiments indicates that there may be more than one mechanism contributing to the uptake of these peptides (1-7, 11, 17-20). Among those mechanisms is the possibility that these peptides may be able to spontaneously translocate across the cell membrane, as suggested by several experiments that indicate a nonendocytotic or energy-independent pathway for the uptake. Because these peptides are highly ...

In addition to endocytosis-mediated cellular uptake, hydrophilic cell-penetrating peptides are able to traverse biological membranes in a non-endocytic mode termed transduction, resulting in immediate bioavailability. Here we analysed structural requirements for the non-endocytic uptake mode of arginine-rich cell-penetrating peptides, by a combination of live-cell microscopy, molecular dynamics simulations and analytical ultracentrifugation. We demonstrate that the transduction efficiency of arginine-rich peptides increases with higher peptide structural rigidity. Consequently, cyclic arginine-rich cell-penetrating peptides showed enhanced cellular uptake kinetics relative to their linear and more flexible counterpart. We propose that guanidinium groups are forced into maximally distant positions by cyclization. This orientation increases membrane contacts leading to enhanced cell penetration.

The delivery of free molecules into the cytoplasm and nucleus by using arginine-rich cell-penetrating peptides (CPPs) has been limited to small cargoes, while large cargoes such as proteins are taken up and trapped in endocytic vesicles. Based on recent work, in which we showed that the transduction efficiency of arginine-rich CPPs can be greatly enhanced by cyclization, the aim was to use cyclic CPPs to transport full-length proteins, in this study green fluorescent protein (GFP), into the cytosol of living cells. Cyclic and linear CPP-GFP conjugates were obtained by using azido-functionalized CPPs and an alkyne-functionalized GFP. Our findings reveal that the cyclic-CPP-GFP conjugates are internalized into live cells with immediate bioavailability in the cytosol and the nucleus, whereas linear CPP analogues do not confer GFP transduction. This technology expands the application of cyclic CPPs to the efficient transport of functional full-length proteins into live cells.

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