X-ray Crystal Structure of a Recombinant Human Myoglobin Mutant at 2-8 A Resolution

• Main Authors: Stevan R. Hubbard, Wayne A. Hendrickson, Howard Hughes, David G. Lambright, Steven G. Boxer
• Other Authors: The Pennsylvania State University CiteSeerX Archives
• Format: Text
• Language: English
• Published: 1989
• Subjects: Kap; Sperm whale
• Online Access: http://citeseerx.ist.psu.edu/viewdoc/summary?doi=10.1.1.538.5003; http://www.stanford.edu/group/boxer/papers/paper74.pdf

We have grown crystals in trigonal space group P3z21 of a mutant human myoglobin, aquomet form, in which lysine at position 45 has been replaced by arginine and cysteine at position 110 has been replaced by alanine. Suitable crystals of native recombinant human myoglobin have not been obtained. We have used the n~olecular replacement method to determine the X-ray crystal structure of the mutant at 2"8 A resolution. At the present s age of refinement, the crystallographic R-value for the model, with tightly restrained stereo-chemistry, is 0.158 for 5"0 to 2"8 A data. As expected, the overall structure is quite similar to the sperm whale myoglobin structure. Arginine 45 adopts a well-ordered conformation similar to that found in aquomet sperm whale myoglobin. We began a crystallographic study of recombi-nant human myoglobin (Mbt) to provide a high-resolution structural basis for site-directed muta-genesis tudies. The aim of such studies is to probe electrostatic nteractions (Varadarajan et al., 1989a) and ligand and proton exchange dynamics. The migration of ligands to the buried heme binding site is of particular interest. Crystallographic evidence suggests that Arg45 (CD3) in sperm whale Mb may be involved in ligand access (Kuriyan et al., 1986). In human Mb, lysine is found at position 45. Whereas one would anticipate that the mutation Lys45 to Arg (K45R) would result in ligand binding Abbreviations used: Mb, myoglobin; K45R, arginine |br lysine replacement a position 45; C110A, alanine for cysteine replacement a position 110; K45R', recombinant human myoglobin with an arginine for lysine replacement a position 45 and an alanine for cysteine replacement a position 110; Kap p, apparent binding constant; 2-D, 2-dimensional; 3-D, 3-dimensional; B-value, isotropic temperature parameter; R-vaiue, standard crystallographic agreement factor; r.m.s., root-mean-square; c.p.u., central processor unit.