Hydrodynamics of horseradish peroxidase revealed by global analysis of multiple fluorescence probes

Abstract

Previous fluorescence studies of horseradish peroxidase conjugated with protoporphyrin IX suggested that the protein behaved hydrodynamically as a prolate ellipsoid of axial ratio 3 to 1. The present study, designed to further investigate the hydrodynamics of this protein, exploits a series of probes, noncovalently bound to the heme binding site of apo-horseradish peroxidase, having different orientations of the excitation and emission transition dipoles with respect to the protein's rotational axes. The probes utilized included protoporphyrin IX and the naphthalene probes 1-anilino-8-naphthalene sulfonate, 2-p-toluidinyl-6-naphthalene sulfonate, and 4,4'-bis(1-anilino-8-naphthalene sulfonate). Time-resolved data were obtained using multifrequency phase fluorometry. The global analysis approach to the determination of molecular shape using multiple probes was evaluated by utilizing all data sets while maintaining a constant molecular shape for the protein. The results indicated that, in such analyses, probes exhibiting a single exponential decay and limited local motion have the major weight in the evaluation of the axial ratio. Probes that show complex decay patterns and local motions, such as the naphthalene derivatives, give rise to significant uncertainties in such global treatments. By explicitly accounting for the effect of such local motion, however, the shape of the protein can be reliably recovered.

Aggregation states of mitochondrial malate dehydrogenase

“…In fact, X-ray crystallographic analysis (Roderick & Banaszak, 1986) has indicated that the mMDH dimer is an ellipsoid with 2:l axial ratio. These value agree, within experimental error, with the "global" rotational relaxation time (103 ns) determined in this study for the FITC-mMDH adduct prepared using protocol P. Moreover, experimentally determined rotational relaxation times on nonspherical molecules depend upon the orientation of the excitation and emission dipoles of the probes with respect to the principal rotational axes of the macromolecule (Beechem et al, 1986: Brunet et al, 1994) and the differences in rotational rates between probes attached at different sites may reflect these orientational differences. Table 1 summarizes studies aimed at determination of the dissociation constant for the dimer/monomer equilibrium of mMDH.…”

Single-Molecule Enzymatic Conformational Dynamics: Spilling Out the Product Molecules

“… 61 , 67 Region 3″ has the longest rotation correlation time, which is close to the rotation correlation time of HRP in solution, 18.3 ns, since the τ r is proportional to the molecular weight and increases about 1 ns for each 2400 Da increase in molecular weight. 68 In region 3″, the resorufin is most likely bound with HRP tightly in the form of an enzyme–product complex, and there is essentially no measurable relative rotation between resorufin and HRP. In regions 2 and 3′, the calculated rotational correlation times are shorter than the rotational correlation time of HRP in the solution.…”

Tubulin equilibrium unfolding followed by time‐resolved fluorescence and fluorescence correlation spectroscopy

“…If the tubulin dimer behaves hydrodynamically as a prolate ellipsoid of axial ratio 2 : 1, one would calculate a rotational correlation time of ∼55 nsec (Weber 1952). In fact, the observed rotational correlation times in such systems will depend upon the orientation of the fluorophore's excitation and emission dipoles with respect to the rotational axes of the ellipsoid (Brunet et al 1994). Hence, our observed rotational correlation times seem reasonable.…”