| Abstract
| - In order to characterize the structural and dynamicfactors that determine the assembly in bhemoproteins, the solution structure of the 98-residue proteinapocytochrome b5 was determined byNMRmethods. Over 800 experimental restraints derived from a series oftwo- and three-dimensional experimentswere used. Holocytochrome b5, the proteinwith iron protoporphyrin-IX liganded to His-39 and His-63,contains in sequence the following elements of secondary structure: β1−α1−β4−β3−α2−α3−β5−α4−α5−β2−α6 [Mathews, F. S., Czerwinski, E. W., & Argos,P. (1979) ThePorphyrins, Vol. 7, pp107−147, Academic Press, New York]. The folded holoproteinpossesses two hydrophobic cores: anextensive, functional core around the heme (core 1), and a smaller,structural core remote from the heme(core 2). The apoprotein was found to contain a stablefour-stranded β-sheet encompassing β1, β2, β3,and β4 and three α-helices, corresponding to α1, α2, and α6.Two short α-helices (α3 and α5) appearto form partially, and α4 is not detected. These three helicesand β5 border the heme binding pocket andare disordered in the apoprotein NMR structure. According tobackbone 1H−15N NOE results, themostflexible region of the apoprotein, except for the termini, extends fromAla-50 (in β5) to Glu-69 (in α5).The polypeptide segment bearing His-63 (located immediately priorto α5) exhibits faster internal motionsthan that bearing His-39 (at the C-terminal end of α2). Thelatter imidazole samples a restricted regionof space, whereas the former can adopt many orientations with respectto the stable core. It was concludedthat heme removal affects the structure and dynamics of most of core 1whereas it leaves core 2 largelyintact. The results provide guidelines for the rational design ofb hemoproteins: a modular structureincluding a packed, stable core and a partially folded binding site isanticipated to present strong kineticand thermodynamic advantages compared to approaches relying on thecomplete formation of secondarystructure prior to heme binding.
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