Characterization of an independent structural unit in apocytochrome b5

Abstract

Apocytochrome b5 is a partially folded protein which contains a stable structural unit under native conditions [Moore, C.D., Al-Misky, O.N., & Lecomte, J.T.J. (1990) Biochemistry 30, 8357-8365]. In this work, the fold of the unit was examined by using 1H and 15N-edited two-dimensional NMR spectroscopy. It was found that it contains four of the five beta-strands and two of the six alpha-helices present in the holoprotein. The remainder of the structure appears to be mostly unstructured and fluctuating among several conformations. The structural unit is stabilized by a hydrophobic core formed by residues from each of the folded elements of secondary structure. Nuclear Overhauser effects and chemical shift values demonstrated that the unit is structurally similar in the apo- and holoproteins. However, the backbone amide hydrogen exchange was found to be much accelerated in the apoprotein. The paramagnetic relaxation agent HyTEMPO was used to probe the packing of the structure. HyTEMPO has unrestricted access to the empty heme binding site whereas it is unable to penetrate the stabilizing core. It was concluded that addition of the heme is necessary for the last strand to dock properly to the rest of the sheet. The kinetics of refolding of the apoprotein were monitored by stopped-flow fluorescence spectroscopy. Extensive protection of the sole tryptophan residue by docking of the two polypeptide termini occurs in less than 60 ms. It was proposed that apocytochrome b5, with its two-region behavior, might serve as a model for the design of proteins which bind a prosthetic group.

A test of the relationship between sequence and structure in proteins: Excision of the heme binding site in apocytochrome b5

“…2B). These contacts can be disrupted by addition of urea, and the results are an increase in Trp fluorescence intensity and a bathochromic shift (Huntley & Strittmatter, 1972;Moore & Lecomte, 1993). Emission spectra in the The line results from global fitting to the three data sets.…”

“…The oxidized holoprotein extinction coefficients were taken to be 114 mM" cm-l at 413 nm (Moore & Lecomte, 1993). The abridged protein and apoprotein extinction coefficients at 280 nm were estimated at 1 1 mM" cm" from the aromatic amino acid content of 1 Trp and 4 Tyr (Mach et al, 1992).…”

“…The two-state unfolding and the observation that the ordered part folds autonomously (Moore & Lecomte, 1993) led to the proposal that apocyt b5 is made of two "modules," one for stability and pre-organization of the second, destined to bind the heme (Falzone et al, 1996). This hypothesis can be tested in various ways.…”

Thermodynamics of apocytochrome b5 unfolding

“…The investigations will be performed on the fragment 1-90. For the tryptic fragment of the holocytochrome bS (from bovine liver), detailed structure is available based on X-ray structure analysis (Mathews et al, 1972), NMR (Moore et al, 1991;Moore & Lecomte, 1993), and calorimetry (Pfeil & Bendzko, 1980).…”

Rapid Mapping of Active Site of KSI by Paramagnetic NMR

“…These paramagnetic perturbations have been used to distinguish the solvent exposed surface area of protein using multidimensional NMR experiments. 10,11 Up till now, however, they have not been intensively applied to the identification of residues located in the active site of enzyme. Δ…”