Solution Structure and Backbone Dynamics of Component IV Glycera dibranchiata Monomeric Hemoglobin-CO †,‡ Brian F. Volkman, § Steve L. Alam, |,⊥ James D. Satterlee, | and John L. Markley* ,§ National Magnetic Resonance Facility at Madison, Department of Biochemistry, UniVersity of WisconsinsMadison, 420 Henry Mall, Madison, Wisconsin 53706, and Department of Chemistry, Washington State UniVersity, Pullman, Washington 99164-4630 ReceiVed April 10, 1998; ReVised Manuscript ReceiVed May 27, 1998 ABSTRACT: The solution structure and backbone dynamics of the recombinant, ferrous CO-ligated form of component IV monomeric hemoglobin from Glycera dibranchiata (GMH4CO) have been characterized by NMR spectroscopy. Distance geometry and simulated annealing calculations utilizing a total of 2550 distance and torsion angle constraints yielded an ensemble of 29 structures with an overall average backbone rmsd of 0.48 Å from the average structure. Differences between the solution structure and a related crystal structure are confined to regions of lower precision in either the NMR or X-ray structure, or in regions where the amino acid sequences differ. 15 N relaxation measurements at 76.0 and 60.8 MHz were analyzed with an extended model-free approach, and revealed low-frequency motions in the vicinity of the heme, concentrated in the F helix. Amide proton protection factors were obtained from H-D amide exchange measurements on 15 N-labeled protein. Patterns in the backbone dynamics and protection factors were shown to correlate with regions of heterogeneity and disorder in the ensemble of NMR structures and with large crystallographic B-factors in the X-ray structures. Surprisingly, while the backbone atoms of the F helix have higher rmsds and larger measures of dynamics on the microsecond to millisecond time scale than the other helices, amide protection factors for residues in the F helix were observed to be similar to those of the other helices. This contrasts with H-D amide exchange measurements on sperm whale myoglobin which indicated low protection for the F helix (S. N. Loh and B. F. Volkman, unpublished results). These results for GMH4 suggest a model in which the F helix undergoes collective motions as a relatively rigid hydrogen-bonded unit, possibly pivoting about a central position near residue Val 87 . The globin family of oxygen-binding heme proteins has been the focus of structure-function analysis by X-ray crystallography, molecular dynamics, and NMR spectroscopy for nearly 40 years (1-3). Interest in these proteins remains high, partly because a precise understanding of the mecha- nism of ligand binding to the heme iron is lacking. Because the heme pocket is effectively inaccessible to solvent, ligand entry must involve some amount of protein structural rearrangement. Time-resolved X-ray crystallography meth- ods have recently been used in an attempt to directly observe the internal motions associated with ligand association- dissociation in sperm whale myoglobin (Mb) 1 (4). Recently developed NMR methods for measuring orientation-depend- ent nuclear dipolar couplings have also been applied to Mb and implicate the H helix in slow collective motions (5). Interest in Glycera dibranchiata monomeric hemoglobins (GMH), a subgroup of the globin superfamily, stems from their unusual ligand binding properties and amino acid substitutions at sequence positions that are highly conserved in other members of the globin family. For example, the E7 Leu found in all GMH proteins replaces the highly conserved globin distal His. Furthermore, sequencing and modeling studies (6) predict that the protein studied here, GMH4, has a B10 Phe, which is normally a Leu in the globin family (7). These hydrophobic side chains create an extremely apolar ligand-binding pocket and may contribute to the altered ligand binding properties of these proteins (8-11). NMR analysis first revealed the reversed orientation † This work was supported by the National Institutes of Health (Grants GM47645 to J.D.S. and GM35976 to J.L.M.). Equipment in the National Magnetic Resonance Facility at Madison (NMRFAM) was purchased with funds from the University of Wisconsin, the NSF Biological Instrumentation Program (Grant DMB-8415048), the NIH Biomedical Research Technology Program (Grant RR02301), the NIH Shared Instrumentation Program (Grant RR02781), and the U.S. Department of Agriculture. Support for the NMR instrumentation at Washington State University was from NIH (Grant RR06312011) and from Battelle Pacific Northwest National Laboratory. ‡ All measured relaxation parameters, calculated model-free param- eters, backbone J coupling constants, H-D amide exchange rates and protection factors, X-PLOR NOE and dihedral constraint files, coor- dinates for the family of 29 conformers, and the refined average structure for GMH4CO have been deposited in the BioMagResBank database, under accession number 4096. Coordinates for the family of 29 conformers (1VRE) and the refined average structure for GMH4CO (1VRF) have been deposited in the Brookhaven Protein Data Bank. * To whom correspondence should be addressed. E-mail: markley@nmrfam.wisc.edu. Phone: (608) 263-9349. Fax: (608) 262- 3759. § University of WisconsinsMadison. | Washington State University. ⊥ Present address: Department of Human Genetics, University of Utah, 15N 2030 E RM 6160, Salt Lake City, UT 84112-5330. 1 Abbreviations: GMH4, component IV G. dibranchiata monomeric hemoglobin; GMH4CO, CO-ligated form of component IV G. dibran- chiata monomeric hemoglobin; Hb, hemoglobin; Mb, myoglobin; HSQC, heteronuclear single-quantum coherence; SE, sensitivity en- hancement; INEPT, insensitive nuclear enhancement by polarization transfer; NOESY, nuclear Overhauser effect spectroscopy. 10906 Biochemistry 1998, 37, 10906-10919 S0006-2960(98)00810-1 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/16/1998