The intrinsically disordered RNR inhibitor Sml1 is a dynamic dimer.

Sml1 is a small ribonucleotide reductase (RNR) regulatory protein in Saccharomyces cerevisiae that binds to and inhibits RNR activation. NMR studies of 15N-labeled Sml1 (104 residues), as well as of a truncated variant (residues 50-104), have allowed characterization of their molecular properties. Sml1 belongs to the class of intrinsically disordered proteins with a high degree of dynamics and very little stable structure. Earlier suggestions for a dimeric structure of Sml1 were confirmed, and from translation diffusion NMR measurements, a dimerization dissociation constant of 0.1 mM at 4 degreesC could be determined. The hydrodynamic radius for the monomeric form of Sml1 was determined to be 23.4 A, corresponding to a protein size between those of a globular protein and a coil. Formation of a dimer results in a hydrodynamic radius of 34.4 A. The observed chemical shifts showed in agreement with previous studies two segments with transient helical structure, residues 4-20 and 60-86, and relaxation studies clearly showed restricted motion in these segments. A spin-label attached to C14 showed long-range interactions with residues 60-70 and 85-95, suggesting that the N-terminal domain folds onto the C-terminal domain. Importantly, protease degradation studies combined with mass spectrometry indicated that the N-terminal domain is degraded before the C-terminal region and thus may serve as a protection against proteolysis of the functionally important C-terminal region. Dimer formation was not associated with significant induction of structure but was found to provide further protection against proteolysis. We propose that this molecular shielding and protection of vital functional structures from degradation by functionally unimportant sites may be a general attribute of other natively disordered proteins.

Differential membrane perturbation caused by the cell penetrating peptide Tp10 depending on attached cargo.

The membrane leakage caused by the cell penetrating peptide Tp10, a variant of transportan, was studied in large unilamellar vesicles with the entrapped fluorophore calcein. The vesicles were composed of zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. A significant decrease in membrane leakage was found when the 55kDa streptavidin protein was attached to Tp10. When a 5.4kDa peptide nucleic acid molecule was attached, the membrane leakage was comparable to that caused by Tp10 alone. The results suggest that direct membrane effects may cause membrane translocation of Tp10 alone and of smaller complexes, whereas these effects do not contribute for larger cargoes.

High cationic charge and bilayer interface-binding helices in a regulatory lipid glycosyltransferase.

In the prokaryote Acholeplasma laidlawii, membrane bilayer properties are sensed and regulated by two interface glycosyltransferases (GTs), synthesizing major nonbilayer- (alMGS GT) and bilayer-prone glucolipids. These enzymes are of similar structure, as many soluble GTs, but are sensitive to lipid charge and curvature stress properties. Multivariate and bioinformatic sequence analyses show that such interface enzymes, in relation to soluble ones of similar fold, are characterized by high cationic charge, certain distances between small and cationic amino acids, and by amphipathic helices. Varying surface contents of Lys/Arg pairs and Trp indicate different membrane-binding subclasses. A predicted potential (cationic) binding helix from alMGS was structurally verified by solution NMR and CD. The helix conformation was induced by a zwitterionic as well as anionic lipid environment, and the peptide was confined to the bilayer interface. Bilayer affinity of the peptide, analyzed by surface plasmon resonance, was higher than that for soluble membrane-seeking proteins/peptides and rose with anionic lipid content. Interface intercalation was supported by phase equilibria in membrane lipid mixtures, analyzed by 31P NMR and DSC. An analogous, potentially binding helix has a similar location in the structurally determined Escherichia coli cell wall precursor GT MurG. These two helices have little sequence conservation in alMGS and MurG homologues but maintain their amphipathic character. The evolutionary modification of the alMGS binding helix and its location close to the acceptor substrate site imply a functional importance in enzyme catalysis, potentially providing a mechanism by which glycolipid synthesis will be sensitive to membrane surface charge and intrinsic curvature strain.

Dynamics of transportan in bicelles is surface charge dependent.

In this study we investigated the dynamic behavior of the chimeric cell-penetrating peptide transportan in membrane-like environments using NMR. Backbone amide 15N spin relaxation was used to investigate the dynamics in two bicelles: neutral DMPC bicelles and partly negatively charged DMPG-containing bicelles. The structure of the peptide as judged from CD and chemical shifts is similar in the two cases. Both the overall motion as well as the local dynamics is, however, different in the two types of bicelles. The overall dynamics of the peptide is significantly slower in the partly negatively charged bicelle environment, as evidenced by longer global correlation times for all measured sites. The local motion, as judged from generalized order parameters, is for all sites in the peptide more restricted when bound to negatively charged bicelles than when bound to neutral bicelles (increase in S2 is on average 0.11 +/- 0.07). The slower dynamics of transportan in charged membrane model systems cause significant line broadening in the proton NMR spectrum, which in certain cases limits the observation of 1H signals for transportan when bound to the membrane. The effect of transportan on DMPC and DHPC motion in zwitterionic bicelles was also investigated, and the motion of both components in the bicelle was found to be affected.

A critical reassessment of penetratin translocation across lipid membranes.

Penetratin is a short, basic cell-penetrating peptide able to induce cellular uptake of a vast variety of large, hydrophilic cargos. We have reassessed the highly controversial issue of direct permeation of the strongly cationic peptide across negatively charged lipid membranes. Confocal laser scanning microscopy on rhodamine-labeled giant vesicles incubated with carboxyfluorescein-labeled penetratin yielded no evidence of transbilayer movement, in contradiction to previously reported results. Confocal fluorescence spectroscopy on black lipid membranes confirmed this finding, which was also not affected by application of a transmembrane electric potential difference. A novel dialysis assay based on tryptophan absorbance and fluorescence spectroscopy demonstrated that the permeability of small and large unilamellar vesicles to penetratin is <10(-13) m/s. Taken together, the results show that penetratin is not capable of overcoming model membrane systems irrespective of the bilayer curvature or the presence of a transmembrane voltage. Thus, direct translocation across the hydrophobic core of the plasma membrane cannot account for the efficient uptake of penetratin into live cells, which is in accord with recent in vitro studies underlining the importance of endocytosis in the internalization process of cationic cell-penetrating peptides.

NMR solution structure and position of transportan in neutral phospholipid bicelles.

Transportan is a chimeric cell-penetrating peptide constructed from the peptides galanin and mastoparan, which has the ability to internalize living cells carrying a hydrophilic load. In this study, we have determined the NMR solution structure and investigated the position of transportan in neutral bicelles. The structure revealed a well-defined alpha-helix in the C-terminal mastoparan part of the peptide and a weaker tendency to form an alpha-helix in the N-terminal domain. The position of the peptide in relation to the membrane, as studied by adding paramagnetic probes, shows that the peptide lies parallel to, and in the head-group region of the membrane surface. This result is supported by amide proton secondary chemical shifts.