Solution (1)H NMR study of the influence of distal hydrogen bonding and N terminus acetylation on the active site electronic and molecular structure of Aplysia limacina cyanomet myoglobin.

The sea hare Aplysia limacina possesses a myoglobin in which a distal H-bond is provided by Arg E10 rather than the common His E7. Solution (1)H NMR studies of the cyanomet complexes of true wild-type (WT), recombinant wild-type (rWT), and the V(E7)H/R(E10)T and V(E7)H mutants of Aplysia Mb designed to mimic the mammalian Mb heme pocket reveal that the distal His in the mutants is rotated out of the heme pocket and is unable to provide a stabilizing H-bond to bound ligand and that WT and rWT differ both in the thermodynamics of heme orientational disorder and in heme contact shift pattern. The mean of the four heme methyl shifts is shown to serve as a sensitive indicator of variations in distal H-bonding among a set of mutant cyanomet globins.

Equilibrium unfolding of a small bacterial cytochrome, cytochrome c551 from Pseudomonas aeruginosa.

The unfolding of the small cytochrome c551 from the bacterium Pseudomonas aeruginosa has been characterized at equilibrium by circular dichroism (CD) and fluorescence spectroscopy. The process can be described by a two state mechanism and the thermodynamic stability of cytochrome c551 is found to be smaller than that of the larger horse cytochrome c (deltaGw = -8.2 vs.

Unfolding of apomyoglobin from Aplysia limacina: the effect of salt and pH on the cooperativity of folding.

The equilibrium unfolding pathway of Aplysia apomyoglobin has been studied under various solvent conditions. The protein exhibits a single unfolding transition in acid in contrast to the two transitions observed for the mammalian apomyoglobins with which it shares a common fold but a low level of sequence identity (24%). This acid-unfolded species has considerable residual structure as evidenced by both tryptophan fluorescence and far-UV CD spectroscopy.

A myoglobin mutant designed to mimic the oxygen-avid Ascaris suum hemoglobin: elucidation of the distal hydrogen bonding network by solution NMR.

The solution 1H NMR structure of the active site and ligand dissociation rate for the cyanomet complex have been determined for a sperm whale myoglobin triple mutant Leu29(B10)-->Tyr, His64(E7)-->Gln, Thr67(E10)-->Arg that mimics the distal residue configuration of the oxygen-avid hemoglobin from Ascaris suum. A double mutant that retains Leu29(B10) was similarly investigated. Two-dimensional NMR analysis of the iron-induced dipolar shifts, together with the conserved proximal side structure for the two mutants, allowed the determination of the orientations of the paramagnetic susceptibility tensor for each complex.

Interactions among residues CD3, E7, E10, and E11 in myoglobins: attempts to simulate the ligand-binding properties of Aplysia myoglobin.

Site-directed mutations have been introduced singly and in combination at residues lysine/arginine45 (CD3), histidine64 (E7), threonine67 (E10), and valine68 (E11) in pig and sperm whale myoglobins. The mutations probe the roles of these key distal pocket residues and represent attempts to mimic the heme environment of Aplysia limacina myoglobin which achieves moderately high O2 affinity in the absence of a distal histidine. In the mollusc myoglobin, arginine-E10 is believed to swing into the heme pocket and provide a hydrogen bond to the bound O2.