Abstract: The interaction of nile red (NR) with apomyoglobin (ApoMb) in the native (pH 7) and molten globule (pH 4) states was investigated using experimental and computational methods. NR binds to hydrophobic locations in ApoMb with higher affinity (K(d) = 25 +/- 5 microM) in the native state than in the molten globule state (K(d) = 52 +/- 5 microM). In the molten globule state, NR is located in a more hydrophobic environment. The dye does not bind to the holoprotein, suggesting that the binding site is located at the heme pocket. In addition to monitoring steady-state properties, the fluorescence emission of NR is capable of tracking submillisecond, time-resolved structural rearrangements of the protein, induced by a nanosecond pH jump. Molecular dynamics simulations were run on ApoMb at neutral pH and at pH 4. The structure obtained for the molten globule state is consistent with the experimentally available structural data. The docking of NR with the crystal structure shows that the ligand binds into the binding pocket of the heme group, with an orientation bringing the planar ring system of NR to overlap with the position of two of the heme porphyrin rings in Mb. The docking of NR with the ApoMb structure at pH 4 shows that the dye binds to the heme pocket with a slightly less favorable binding energy, in keeping with the experimental K(d) value. Under these conditions, NR is positioned in a different orientation, reaching a more hydrophobic environment in agreement with the spectroscopic data.

Abstract: The acidic transition of porcine plasminogen, prepared by affinity chromatography, was studied by non-destructive methods. These methods are based on the analysis of the behaviour of the tryptophyls under various conditions. The perturbation of the absorption and emission spectra by pH or temperature and the dynamic quenching of the intrinsic fluorescence are used to obtain information on structural changes which affect the environment of these residues. It is shown that by decreasing pH the fluorescence emission spectra are shifted toward the long wavelengths, with a broadening of the fluorescence band. The same effect can be obtained at constant pH by heating the protein solution. In order to analyze these phenomena, it is assumed that the fluorescence intensities at 355 nm and 328 nm reflect the proportion of the tryptophans which are exposed to the solvent, and buried, respectively. The plot of the ratio of the fluorescence intensities at these wavelengths versus pH or temperature leads to a titration curve showing an unmasking of tryptophans. The proportion of exposed tryptophans is measured by the dynamic fluorescence quenching technique and the data analyzed according to Lehrer. The plot of the fraction of exposed tryptophyls versus pH also shows the unmasking of these chromophores. Thermal perturbation of a solution of plaminogen at neutral pH induces a difference absorption spectrum whose amplitudes at the maxima are proportional to the number of exposed aromatic residues. The comparison with a solution of fully denatured plasminogen in 6 M guanidium chloride, where all the tryptophyls are exposed, shows that the percentage of exposure is equal to 59%. This number is significantly higher than the percentage found by the fluorescence quenching technique (20%), indicating that some tryptophyls are located in crevices, exposed to the solvent but not to the iodide. At acidic pH the absorption difference spectra induced by thermal perturbation are not classical, since they show an inversion and a new band between 300 nm and 305 nm. This band is mentioned in the literature as a minor band of tryptophan which appears when this chromophore is located in an asymmetric environment. On plotting the maximum amplitude of these spectra obtained at acidic pH versus temperature, we obtain a curve indicating that two types of antagonistic interactions are involved in the perturbation of the chromophores spectra. The spectrophotometric titration of plasminogen gives classical absorption difference spectra. By plotting the maximum amplitude at 292 nm versus pH, we obtain a titration curve with an apparent pK of 2.9 units. This pK is acidic which respect to the pK value of a normal carboxyl. This low value can be due to a positively charged group in the neighbourhood of a carboxyl, which interacts with one or more chromophores. When the carboxyl becomes protonated, this positively charged group is free and available to perturb the environment of some chromophores...

Abstract: The effect of His-heme misligation on folding has been investigated for a triple mutant of yeast iso-2 cytochrome c (N26H,H33N,H39K iso-2). The variant contains a single misligating His residue at position 26, a location at which His residues are found in several cytochrome c homologues, including horse, tuna, and yeast iso-1. The amplitude for fast phase folding exhibits a strong initial pH dependence. For GdnHCl unfolded protein at an initial pH<5, the observed refolding at final pH 6 is dominated by a fast phase (tau(2f)=20 ms, alpha(2f)=90 %) that represents folding in the absence of misligation. For unfolded protein at initial pH 6, folding at final pH 6 occurs in a fast phase of reduced amplitude (alpha(2f) approximately 20 %) but the same rate (tau(2f)=20 ms), and in two slower phases (tau(m)=6-8 seconds, alpha(m) approximately 45 %; and tau(1b)=16-20 seconds, alpha(1b) approximately 35 %). Double jump experiments show that the initial pH dependence of the folding amplitudes results from a slow pH-dependent equilibrium between fast and slow folding species present in the unfolded protein. The slow equilibrium arises from coupling of the His protonation equilibrium to His-heme misligation and proline isomerization. Specifically, Pro25 is predominantly in trans in the unligated low-pH unfolded protein, but is constrained in a non-native cis isomerization state by His26-heme misligation near neutral pH. Refolding from the misligated unfolded form proceeds slowly due to the large energetic barrier required for proline isomerization and displacement of the misligated His26-heme ligand.