[en] The structure and dynamics of the Pf1 and fd bacteriophage coat proteins in detergent micelles are characterized in solution by nuclear magnetic resonance spectroscopy. The coat proteins are found to exist within the bacterial inner cell membrane during viral infection and assembly. The coat proteins serve as a model system to investigate integral membrane proteins as well as the viral infection and assembly processes. The coat protein is insoluble in aqueous or organic solvents and can only be effectively solubilized in the presence of detergents that form micelles or phospholipids that form vesicles. The effective molecular weight of the detergent-micelle complex is ca. 30K daltons. Sequential assignment strategies were ineffective due to short T/sub 2s/ and severe resonance degeneracy. The backbone resonance assignments were completed by the combination of several homo- and heteronuclear correlation techniques with biosynthetic ¹⁵N labelling. 2D NOE experiments were used to locate and characterize the secondary structure of the membrane bound form of the proteins showing them to be largely helical with the hydrophobic core existing in a very stable helix

[en] Published in summary form only

[en] Human urinary-type plasminogen activator (urokinase) and proteolytic fragments corresponding to the kringle, EGF-kringle, and protease domains have been examined by ¹H NMR spectroscopy. The intact protein shows a very well-resolved spectrum for a molecule of this size (MW 54,000), with resonance line widths not greatly increased from those of the isolated domains. On increasing the temperature, the protein at pH values close to 4 was found to undergo two distinct and reversible conformational transitions. These were identified, by comparison with spectra of the proteolytic fragments, as the unfolding of the kringle (and EGF) domains (at ∼ 42 degree C) and of a segment of the protease domain (at ∼ 60 degree C). The remaining segment of the protease domain showed persistent structure to at least 85 degree C at pH 4; only at lower pH values could complete unfolding be achieved. The results indicate that the structures and stabilities of the isolated domains are closely similar to those in the intact protein and suggest that there is a degree of independent motion at least between the kringle and protease domains

[en] The major coat proteins of the fd (M13) and Pf1 filamentous bacteriophage exist as integral membrane proteins during the viral life cycle. These proteins adopt their membrane bound conformations when solubilized by a variety of detergents, and the protein-detergent micelle complexes can be studied using solution NMR techniques. Determination of the structure of the coat proteins in their membrane bound form has been accomplished by qualitative interpretation of 2-dimensional ¹H-¹H NOE spectra (NOESY). The critical amide proton resonance assignments were made through biosynthetic ¹⁵N labeling and ¹H/¹⁵N heteronuclear chemical shift correlation techniques. The data indicate that both proteins adopt helical conformations within the micelle. The ¹⁵N/¹H heteronuclear NOE has been used to characterize the backbone dynamics of both proteins in micelles. The lipid associated residues of the proteins are rigid on the nanosecond timescale, while the hydrophilic solvent associated N- and C-termini are high mobile. These results complement previously reported protein dynamics studies of membrane bound coat proteins conducted using solid state NMR methods. Solid state NMR studies reported in the literature have also investigated the structure and dynamics of the fd and Pf1 major coat proteins when bound to intact phage. Therefore, structure/dynamics comparisons of the proteins in their structural versus membrane bound forms can be made

[en] The major coat protein of filamentous bacteriophage adopts its membrane-bound conformation in detergent micelles. High-resolution ¹H and ¹⁵N NMR experiments are used to characterize the structure and dynamics of residues 30-40 in the hydrophobic midsection of Pf1 coat protein in sodium dodecyl sulfate micelles. Uniform and specific-site ¹⁵N labels enable the immobile backbone sites to be identified by their ¹H/¹⁵N heteronuclear nuclear Overhauser effect and allow the assignment of ¹H and ¹⁵N resonances. About one-third of the amide N-H protons in the protein undergo very slow exchange with solvent deuterons, which is indicative of sites in highly structured environments. The combination of results from ¹H/¹⁵N heteronuclear correlation, ¹H homonuclear correlation, and ¹H homonuclear Overhauser effect experiments assigns the resonances to specific residues and demonstrates that residues 30-40 of the coat protein have a helical secondary structure

[en] Published in summary form only