Mapping the orientation of helices in micelle-bound peptides by paramagnetic relaxation waves.

Many antimicrobial peptides form alpha-helices when bound to a membrane. In addition, around 80% of residues in membrane-bound proteins are found in alpha-helical regions. The orientation and location of such helical peptides and proteins in the membrane are key factors determining their function and activity. Here we present a new solution state NMR method for obtaining the orientation of helical peptides in a membrane-mimetic environment (micelle-bound) without any chemical perturbation of the peptide-micelle system. By monitoring proton longitudinal relaxation rates upon addition of the freely water-soluble and inert paramagnetic probe Gd(DTPA-BMA) to an alpha-helical peptide, a wavelike pattern with a periodicity of 3.6 residues per turn is observed. The tilt and azimuth (rotation) angle of the helix determine the shape of this paramagnetic relaxation wave and can be obtained by least-square fitting of measured relaxation enhancements. Results are presented for the 15-residue antimicrobial peptide CM15 which forms an amphipathic helix almost parallel to the surface of the micelle. Thus, a few fast experiments enable the identification of helical regions and determination of the helix orientation within the micelle without the need for covalent modification, isotopic labeling, or sophisticated equipment. This approach opens a path toward the topology determination of alpha-helical membrane-proteins without the need for a complete NOE-based structure determination.

Functional characterization of Glu298Asp mutant human endothelial nitric oxide synthase purified from a yeast expression system.

The Glu298Asp polymorphism of human endothelial nitric oxide synthase (eNOS) has been reported to be associated with several cardiovascular diseases, including hypertension and myocardial infarction. Therefore, we investigated the effect of the Glu298Asp (E298D) mutation on the function of purified recombinant eNOS expressed in the yeast Pichia pastoris. Wild type (WT) and mutant exhibited comparable affinities for L-arginine (K(m) values 4.4+/-0.6 and 5.2+/-0.8 microM, respectively) and V(max) values (142+/-36 and 159+/-29 nmol of L-citrulline/mg min, respectively). The E298D mutation affected neither electron transfer through the reductase domain (measured as cytochrome c reduction) nor reductive O(2) activation (measured either as NADPH oxidation or as H(2)O(2) formation in the absence of L-arginine and tetrahydrobiopterin (BH4)). The mutant was activated by BH4 with an EC(50) of 0.24+/-0.04 microM, a value comparable to that obtained with WT eNOS (0.22+/-0.02 microM). Activation of the enzyme by Ca(2+) was not affected (EC(50)=0.50+/-0.04 and 0.49+/-0.02 microM for WT and E298D eNOS, respectively). Calmodulin (CaM) affinity, studied by radioligand binding using 125I-labeled CaM, revealed virtually identical K(D) (3.2+/-0.5 and 4.0+/-0.3nM) and B(max) (1.4+/-0.2 and 1.2+/-0.3 pmol/pmol subunit) values for WT and E298D eNOS, respectively. Furthermore, E298D eNOS did not differ from the WT enzyme with respect to heme and flavin content or the ability to form SDS-resistant dimers. To summarize, we obtained no evidence for altered enzyme function of the eNOS mutant that could explain endothelial dysfunction associated with the E298D polymorphism.