SOLUTION STRUCTURE OF CARBONMONOXY MYOGLOBIN DETERMINED FROM NUCLEAR-MAGNETIC-RESONANCE DISTANCE AND CHEMICAL-SHIFT CONSTRAINTS

Solution NMR structures for sperm whale carbonmonoxy myoglobin have be en calculated using 1301 distance restraints determined from nuclear O verhauser enhancement (NOE) measurements on N-15-labeled protein and c hemical shift calculations for 385 protons. Starting structures includ ed four crystal forms of myoglobin and 12 structures generated by metr ic matrix distance geometry. Refinements were also carried out using d istance restraints alone. In general, the solution conformations are v ery close to the crystal structures, although the crystal structures a re not consistent with some of the observed NOE connectivities. The so lution structures are about as far apart from each other (as measured by backbone root-mean-square deviations) as they are from the crystal conformation. Inclusion of chemical shift restraints both tightened th e spread of computed structures (especially in the heme pocket region) and led to structures that were closer to the X-ray conformation. The disposition of the side-chains near the heme group could in many case s be determined with considerable confidence, suggesting that a chemic al shift analysis may be a useful adjunct to other sources of structur al information available from NMR. In particular, this evidence sugges ts that the distal histidine residue is slightly displaced from the cr ystal conformation, but still inside the heme pocket at pH 5.6, that t he side-chain of Leu89 is in contact with the heme ring but is probabl y disordered, and that the heme pocket where ligands bind is virtually identical in solution and in the crystal forms.