Recently, much attention has been given to the problem of drug delivery through the cell-membrane in order to treat and manage several diseases. The discovery of cell penetrating peptides (CPPs) represents a major breakthrough for the transport of large-cargo molecules that may be useful in clinical applications. CPPs are rich in basic amino acids such as arginine and lysine and are able to translocate over membranes and gain access to the cell interior. They can deliver large-cargo molecules, such as oligonucleotides, into cells. Endocytosis and direct penetration have been suggested as the two major uptake mechanisms, a subject still under debate. Unresolved questions include the detailed molecular uptake mechanism(s), reasons for cell toxicity, and the delivery efficiency of CPPs for different cargoes. Here, we give a review focused on uptake mechanisms used by CPPs for membrane translocation and certain experimental factors that affect the mechanism(s).

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Metal binding to the amyloid β‐peptide is suggested to be involved in the pathogenesis of Alzheimer's disease. We used high‐resolution NMR to study zinc binding to amyloid β‐peptide 1–40 at physiologic pH. Metal binding induces a structural change in the peptide, which is in chemical exchange on an intermediate rate, between the apo‐form and the holo‐form, with respect to the NMR timescale. This causes loss of NMR signals in the resonances affected by the binding. Heteronuclear correlation experiments, 15N‐relaxation and amide proton exchange experiments on amyloid β‐peptide 1–40 revealed that zinc binding involves the three histidines (residues 6, 13 and 14) and the N‐terminus, similar to a previously proposed copper‐binding site [Syme CD, Nadal RC, Rigby SE, Viles JH (2004) J Biol Chem279, 18169–18177]. Fluorescence experiments show that zinc shares a common binding site with copper and that the metals have similar affinities for amyloid β‐peptide. The dissociation constant Kd of zinc for the fragment amyloid β‐peptide 1–28 was measured by fluorescence, using competitive binding studies, and that for amyloid β‐peptide 1–40 was measured by NMR. Both methods gave Kd values in the micromolar range at pH 7.2 and 286 K. Zinc also has a second, weaker binding site involving residues between 23 and 28. At high metal ion concentrations, the metal‐induced aggregation should mainly have an electrostatic origin from decreased repulsion between peptides. At low metal ion concentrations, on the other hand, the metal‐induced structure of the peptide counteracts aggregation.

One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.

Peptide nucleic acid (PNA) is a DNA analogue in which the negatively charged sugar phosphate backbone has been substituted by uncharged N-(2-aminoethyl)glycine units. The study of a PNA−DNA duplex and the corresponding DNA−DNA duplex gives a unique opportunity to compare two polyelectrolytes with virtually identical geometry but greatly different linear charge density. The results provide a basis for a study of the applicability of the Poisson−Boltzmann (PB) and counterion condensation (CC) theories. UV and circular dichroism spectroscopy as well as isothermal titration calorimetry (ITC) have been used to study the effect of different ions on the stability and conformation of PNA−DNA, PNA−PNA, and DNA−DNA duplexes having the same base sequences. Cations in general destabilize both antiparallel (N/3‘) and parallel (N/5‘) PNA−DNA duplexes whereas they stabilize the DNA−DNA duplex. Studies on the effect of monovalent salt such as NaCl on T m were carried out over a wide range of salt concentrations (0.01 to 5 M). The decrease in the T m of the N/3‘ PNA−DNA duplex with increasing ionic strength in the range of concentrations of 0.01 to 0.5 M, where electrostatic effects predominate, is explained in terms of counterion release upon duplex formation in contrast to the counterion association accompanying the formation of a DNA duplex. The uncharged PNA−PNA duplex shows no significant destabilization in this concentration range. The higher stability of the N/3‘ PNA−DNA compared to the DNA−DNA duplex (ΔΔG ∼ −7 kcal/mol) is ascribed to more favorable entropic contributions consistent with the counterion release that accompanies the PNA−DNA duplex formation. At high salt concentration (>1 M), where electrostatic contributions saturate, similar trends in the decrease in T m were observed for the three types of duplexes irrespective of their backbone charges. The destabilizing effects of a series of Na salts with various monovalent anions on N/3‘ PNA−DNA and PNA−PNA duplexes were found to follow the Hofmeister series, emphasizing the importance of the hydrophobic interaction between nucleobases for the stability of the PNA complexes in high salt concentration.

Although metallocofactors are ubiquitous in enzyme catalysis, how metal binding specificity arises remains poorly understood, especially in the case of metals with similar primary ligand preferences such as manganese and iron. The biochemical selection of manganese over iron presents a particularly intricate problem because manganese is generally present in cells at a lower concentration than iron, while also having a lower predicted complex stability according to the Irving-Williams series (Mn II < Fe II < Ni II < Co II < Cu II > Zn II ). Here we show that a heterodinuclear Mn/Fe cofactor with the same primary protein ligands in both metal sites self-assembles from Mn II and Fe II in vitro, thus diverging from the Irving-Williams series without requiring auxiliary factors such as metallochaperones. Crystallographic, spectroscopic, and computational data demonstrate that one of the two metal sites preferentially binds Fe II over Mn II as expected, whereas the other site is nonspecific, binding equal amounts of both metals in the absence of oxygen. Oxygen exposure results in further accumulation of the Mn/Fe cofactor, indicating that cofactor assembly is at least a twostep process governed by both the intrinsic metal specificity of the protein scaffold and additional effects exerted during oxygen binding or activation. We further show that the mixed-metal cofactor catalyzes a two-electron oxidation of the protein scaffold, yielding a tyrosine-valine ether cross-link. Theoretical modeling of the reaction by density functional theory suggests a multistep mechanism including a valyl radical intermediate.H alf of all enzymes are estimated to contain metallocofactors (1). An important subset uses transition metal ions to perform key redox reactions such as oxygen activation. The diiron cofactor of the ferritin-like superfamily of proteins is particularly versatile (2). While ferritin itself simply oxidizes and sequesters iron (3), in other family members the diiron center acts as a transient one-or two-electron oxidant. In the R2 subunits of class I ribonucleotide reductases (RNRs) it generates a redoxactive tyrosyl radical (4, 5), whereas in the bacterial multicomponent monooxygenases (BMMs) it catalyzes the hydroxylation of a variety of hydrocarbons (6). For four decades it was assumed that all ferritin superfamily proteins contained diiron cofactors. However, in recent years new subfamilies harboring either a dimanganese or heterodinuclear Mn/Fe cofactor have been documented (7)(8)(9)(10)(11)(12)(13)(14). The Mn/Fe cofactor was discovered in class Ic RNR R2 subunits, where its Mn IV /Fe III state functionally replaces the diiron-tyrosyl radical cofactor of class Ia R2s (9, 10). After a long controversy, class Ib R2 proteins were shown to use a dimanganese cofactor in the same scaffold (7,8). These recent developments highlight the complexity of correctly identifying the metals that make up native metallocofactors. While the metal preferences of some primary coordination motifs are well known and distinct, others ar...

Ribonucleotide reductase (RNR) synthesizes the deoxyribonucleotides for DNA synthesis. The R2 protein of normal class I ribonucleotide reductases contains a diiron site that produces a stable tyrosyl free radical, essential for enzymatic activity. Structural and electron paramagnetic resonance studies of R2 from Chlamydia trachomatis reveal a protein lacking a tyrosyl radical site. Instead, the protein yields an iron-coupled radical upon reconstitution. The coordinating structure of the diiron site is similar to that of diiron oxidases/monoxygenases and supports a role for this radical in the RNR mechanism. The specific ligand pattern in the C. trachomatis R2 metal site characterizes a new group of R2 proteins that so far has been found in eight organisms, three of which are human pathogens.

The self-complementary DNA decamer duplex d(CTGAATTCAG)2 and its modified counterpart d(CTGA[2AP]TTCAG)2, where the innermost adenine (6-aminopurine) has been replaced with the fluorescent analogue 2-aminopurine (2AP), have been studied by fluorescence and NMR spectroscopy and simulated by molecular dynamics. Both decamers are recognized and cleaved by the EcoRI restriction endonuclease. 2D NMR results show that both decamers have a standard B-type conformation below 20 degrees C, though a disturbance exists to the 5' side of the 2AP site which may originate from increased local mobility. The fluorescence and fluorescence anisotropy decays of both decamers, as well as the one containing 2AP in only one chain, were studied as a function of temperature. The data show that the 2AP base exists in a temperature-dependent distribution of states and shows rapid motions, suggesting interconversion among these states on a time scale of about 10(-10) s. The integrated fluorescence of the decamer with 2AP in both chains shows a large increase around the helix melting temperature whereas the decamer with one 2AP shows only a mild increase, showing that the mixed helix has a different structural transition as sensed by the 2AP base. The data suggest a model of conformational states which have distinct fluorescence decay times. The various states may differ in the degree of base stacking. Fluctuations in the degree of stacking of the A or 2AP base are supported by molecular dynamics simulations, which additionally show that the 2AP-T or A-T base pair hydrogen bonds remain intact during these large motions.