The antioxidant activity of rosemary (Rosmarinus officinalis L.) extracts is mainly due to phenolic abietane diterpenes and phenolic acids such as rosmarinic acid. In this study a comprehensive characterization of non-water-soluble and water-soluble extracts from rosemary was achieved by liquid chromatography coupled to electrospray and mass spectrometry. The antioxidant activity of these extracts and their respective major compounds (carnosic acid, carnosol, rosmadial, genkwanin, and rosmarinic acid) was analyzed and compared by using different in vitro systems. Whereas rosmarinic acid, carnosic acid, and carnosol exhibited similar antioxidant activity in a phospholipid membrane-free assay, carnosol behaved as an extremely potent antioxidant in a membrane-based assay (4-6 times stronger than the rest of the compounds). This differential antioxidant behavior suggests that factors other than the radical scavenging capability may be involved. All of the diterpenes induced severe effects on lipid order and packing of phospholipid model membranes. Rosmadial and carnosol decreased the number and/or mobility of water molecules located at the polar head group region of the membrane phospholipids as seen by Laurdan fluorescence spectroscopy. Carnosol also strongly enhanced lipid order at the hydrophobic core of the membrane. These effects throughout the bilayer correlated to the stronger antioxidant capacity of carnosol to inhibit lipid peroxidation. On the contrary, carnosic acid decreased membrane fluidity at deeper regions of the bilayer as measured by bilayer-to-micelle transition assay and self-quenching measurements by using octadecylrhodamine B. These effects may contribute to membrane stabilization and hindrance of radical propagation, which may cooperate with the electron donor ability of rosemary diterpenes in protecting the membranes against oxidative damage.

The type 1 HIV presents a conical capsid formed by ∼1500 units of the capsid protein, CA. Homodimerization of CA via its C-terminal domain, CA-C, constitutes a key step in virion assembly. CA-C dimerization is largely mediated by reciprocal interactions between residues of its second ␣-helix. Here, we show that an N-terminal-acetylated and C-terminal-amidated peptide, CAC1, comprising the sequence of the CA-C dimerization helix plus three flanking residues at each side, is able to form a complex with the entire CA-C domain. Thermal denaturation measurements followed by circular dichroism (CD), NMR, and size-exclusion chromatography provided evidence of the interaction between CAC1 and CA-C. The apparent dissociation constant of the heterocomplex formed by CA-C and CAC1 was determined by several biophysical techniques, namely, fluorescence (using an anthraniloyl-labeled peptide), affinity chromatography, and isothermal titration calorimetry. The three techniques yielded similar values for the apparent dissociation constant, in the order of 50 M. This apparent dissociation constant was only five times higher than was the dissociation constant of both CA-C and the intact capsid protein homodimers (10 M).

The sterile alpha motif (SAM) domain is a protein module of approximately 65 to 70 amino acids found in many diverse proteins whose functions range from signal transduction to transcriptional repression. The alpha splice variant of p73 (p73 alpha), a homologue of the tumor suppressor p53, has close to its C-terminus a SAM motif. Here, we report the folding equilibrium properties of the p73 alpha SAM domain (SAMp73) by using different biophysical techniques (circular dichroism, fluorescence, and Fourier transform infrared spectroscopies, and differential scanning calorimetry). Those probes indicate that SAMp73 folds via a two-state mechanism. Fluorescence experiments performed at different pHs showed two titrations: the first one due to an acid residue (with a pK(a) = 4.5 +/- 0.3) and the second due to deprotonation of tyrosine residues. The conformational stability of the protein upon chemical denaturation was determined over the pH range 3 to 10. The maximum conformational stability is DeltaG = 5.7 +/- 0.4 kcal x mol(-1) (at 25 degrees C) and occurs in a broad maximum, with little variation, between pH 6 and 10. The high melting temperature of SAMp73 (T(m) = 93.5 degrees C), despite its moderate conformational stability at 25 degrees C, can be ascribed to its low heat capacity change upon unfolding, DeltaC(p), which is estimated to be around 915 cal x K(-1) x mol(-1) at 25 degrees C and only around 543 cal x K(-1) x mol(-1) at the T(m). The implications of the temperature-dependent nature of DeltaC(p) are discussed in relation to the thermal stability of proteins as opposed to their conformational stability at room temperature.

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