Magdalena Svensson

PROPERTIES OF SPIN AND FLUORESCENT LABELS AT A RECEPTOR-LIGAND INTERFACE

Site-directed labeling was used to obtain local information on the binding interface in a receptor-ligand complex. As a model we have chosen the specific association of the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa), the primary initiator of the blood coagulation cascade. Different spectroscopic labels were covalently attached to an engineered cysteine in position 140 in sTF, a position normally occupied by a Phe residue previously characterized as an important contributor to the sTF:FVIIa interaction. Two spin labels, IPSL [N-(1-oxyl-2,2,5, 5-tetramethyl-3-pyrrolidinyl)iodoacetamide] and MTSSL [(1-oxyl-2,2,5, 5-tetramethylpyrroline-3-methyl)methanethiosulfonate], and two fluorescent labels, IAEDANS [5-((((2-iodoacetyl)amino) ethyl)amino)naphthalene-1-sulfonic acid] and BADAN [6-bromoacetyl-2-dimethylaminonaphthalene], were used. Spectral data from electron paramagnetic resonance (EPR) and fluorescence spectroscopy showed a substantial change in the local environment of all labels when the sTF:FVIIa complex was formed. However, the interaction was probed differently by each label and these differences in spectral appearance could be attributed to differences in label properties such as size, polarity, and/or flexibility. Accordingly, molecular modeling data suggest that the most favorable orientations are unique for each label. Furthermore, line-shape simulations of EPR spectra and calculations based on fluorescence depolarization measurements provided additional details of the local environment of the labels, thereby confirming a tight protein-protein interaction between FVIIa and sTF when the complex is formed. The tightness of this local interaction is similar to that seen in the interior of globular proteins.

Cis‐trans isomerization is rate‐determining in the reactivation of denatured human carbonic anhydrase II as evidenced by proline isomerase

The refolding of human carbonic anhydrase II is a sequential process. The slowest step involved is the recovery of enzymic activity (t½=9 min). Kinetic data from ‘double‐jump’ measurements indicate that proline isomerization might be rate determining, in the reactivation of the denatured enzyme. Proof of this is provided by the effect of proline isomerase on the reactivation kinetics; the presence of isomerase during reactivation lowers the half‐time or the reaction to 4 min, and inhibition of proline isomerase completely abolishes this kinetic effect. A similar acceleration of the refolding process by proline isomerase is also observed for bovine carbonic anhydrase II, in contrast to what has previously been reported. In human carbonic anhydrase II there are two cis‐peptidyl‐Pro bonds at Pro30 and Pro202. Two asparagine single mutants (P30N and P202N) and a glycine double mutant (P30G/P202G) wore constructed to investigate the role of these prolines in the rate limitation of the reactivation process. Both in the presence and absence of PPlase the P202N mutant behaved exactly like the unmutaled enzyme, Thus, cis‐trans isomerization of the Pro202 cis‐peptidyl bond is not rate determining in the reactivation process, The mutations at position 30 led to such extensive destabilization of the protein that the refolding reaction could not be studied.

Characterization of a folding intermediate of human carbonic anhydrase II: probing local mobility by electron paramagnetic resonance

The spin-labeling method was used to investigate human carbonic anhydrase, HCA II, undergoing unfolding induced by guanidine-HCI (Gu-HCI). The spin-probe, N-(2,2,5,5-tetramethyl-1-yloxypyrrolidinyl-3-yl)iodoacetamide, was attached covalently to the single cysteine (position 206) in the enzyme. The electron paramagnetic resonance spectrum of the folded structure showed the characteristic slow motional spectra. When the concentration of the denaturing agent, Gu-HCI, was gradually increased, new spectral components with narrower lines evolved to give complex electron paramagnetic resonance spectra, apparently containing superimposed contributions from several components of different mobility. By a differentiation technique, it was possible to follow the relative increase of the narrow components as a function of Gu-HCI concentration. The amplitude of difference spectra versus Gu-HCI concentration showed two distinct maxima, indicating the existence of a folding intermediate state/structure. The results were found to agree with optical absorption data, which showed similar transitions at the same Gu-HCI concentrations. From line-shape simulations assuming a Brownian diffusion model, the rotational diffusion constants for the spin-label in the folded, folding intermediate, and unfolded structures were determined. The relative abundances of the three conformations in the region 0-4 M Gu-HCI were obtained by least squares fitting of the simulated spectra to the experimental ones. The folding intermediate was found to have a maximum population of 39 +/- 4% at approximately 0.7 M Gu-HCI.

Spin and fluorescent probing of the binding interface between tissue factor and factor VIIa at multiple sites.

The specific complex between the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa) was chosen as a model for studies of the binding interface between two interacting proteins. Six surface-exposed positions in sTF, residues known to contribute to the sTF-FVIIa interaction, were selected for cysteine mutation and site-directed labeling with spin and fluorescent probes. The binding interface was characterized by spectral data from electron paramagnetic resonance (EPR) and steady-state and time-domain fluorescence spectroscopy. The labels reported on compact local environments at positions 158 and 207 in the interface region between sTF and the gamma-carboxyglutamic acid (Gla) domain of FVIIa, and at positions 22 and 140 in the interface region between sTF and the first epidermal growth factor-like (EGF1) domain of FVIIa. The tightness of the local interactions in these parts of the interface is similar to that seen in the interior of globular proteins. This was further emphasized by the reduced local polarity detected by the fluorescent label upon FVIIa binding, especially in the sTF-Gla region. There were indications of structural rigidity also at positions 45 and 94 in the interface region between sTF and the protease domain (PD) of FVIIa, despite the perturbed cofactor function of these sTF variants. The results of the present study indicate that the multi-probing approach enables comparison of the tightness and characteristics of interaction along the binding interface of a protein complex. This approach also increases the probability of acquiring reliable structural data that are descriptive of the wild-type proteins.

Probing local mobility in carbonic anhydrase: EPR of spin-labelled SH groups introduced by site-directed mutagenesis

Cloned human carbonic anhydrase, HCAII, and mutants thereof have been investigated by spin-probing methods during its unfolding caused by guanidine·HCl. The spin-probe, N-(2,2,5,5-tetramethyl-1-ylooxypyrrolidin-3-yl)iodoacetamide, has been regiospecifically introduced into cyst-eines by site-directed mutagenesis in various positions of the protein structure. Here we focus on EPR spectra of three different spin-labelled enzymes at various guanidine-HCl concentrations (at equilibrium). The following spin-labelled mutants are discussed: W16C/C206S, W97C/C206S and F176C/C206S. The EPR spectra of the three mutants differ markedly at low guanidine·HCl concentration (0–1 mol dm–3) particularly within the series W97C/C206S and F176C/C206S, showing the characteristic anisotropic slow motional features. The W16C/C206S label position is much more mobile in the folded structures. The rotational correlation times reflect the local environment of the spin-probe in the folded enzyme: in W97C it is located in the core, near the active centre, in W16C and F176C at more peripheral positions. All samples gave EPR spectra characteristic of a ‘free’ unfolded protein chain at guanidine-HCl concentrations of ca. 3 mol dm–3 and above, and could be characterised by using one component in lineshape simulations. The spectra of the W97C/C206S and F176C/C206S samples in low concentrations of guanidine·HCl (between ca. 0.1 and 2.0 mol dm–3) could only be reproduced in simulations by introducing several components associated with rather different rotational correlation times. This seems to imply the co-existence of at least two dynamic structures in equilibrium during the intermediate stages of the unfolding process. It is compatible with earlier suggestions of a folding intermediate based on optical characterisation.

Site-directed fluorescence probing to dissect the calcium-dependent association between soluble tissue factor and factor VIIa domains

We have used the site-directed labeling approach to study the Ca(2+)-dependent docking of factor VIIa (FVIIa) to soluble tissue factor (sTF). Nine Ca(2+) binding sites are located in FVIIa and even though their contribution to the overall binding between TF and FVIIa has been thoroughly studied, their importance for local protein-protein interactions within the complex has not been determined. Specifically we have monitored the association of the gamma-carboxyglutamic acid (Gla), the first EGF-like (EGF1), and the protease domains (PD) of FVIIa to sTF. Our results revealed that complex formation between sTF and FVIIa during Ca(2+) titration is initiated upon Ca(2+) binding to EGF1, the domain containing the site of highest Ca(2+) affinity. Besides we showed that a Ca(2+)-loaded Gla domain is required for an optimal association of all domains of FVIIa to sTF. Ca(2+) binding to the PD seems to be of some importance for the docking of this domain to sTF.

Effects on the conformation of FVIIa by sTF and Ca(2+) binding: Studies of fluorescence resonance energy transfer and quenching

The apparent length of FVIIa in solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein (Fl-FPR) and a rhodamine derivative (TMR), were covalently attached to FVIIa. The binding site of Fl-FPR was in the protease domain whereas TMR was positioned in the Gla domain, thus allowing a length measure over virtually the whole extension of the protein. From the FRET measurements, the distances between the two probes were determined to be 61.4 for free FVIIa and 65.5Å for FVIIa bound to soluble tissue factor (sTF). These seemingly short distances, compared to those anticipated based on the complex crystal structure, require that the probes stretch towards each other. Thus, the apparent distance from the FRET analysis was shown to increase with 4Å upon formation of a complex with sTF in solution. However, considering how protein dynamics, based on recent molecular dynamics simulations of FVIIa and sTF:FVIIa (Y.Z. Ohkubo, J.H. Morrissey, E. Tajkhorshid, J. Thromb. Haemost. 8 (2010) 1044-1053), can influence the apparent fluorescence signal our calculations indicated that the global average conformation of active-site inhibited FVIIa is nearly unaltered upon ligation to sTF. It is known from amidolytic activity measurements that Ca(2+) binding leads to activation of FVIIa, but we have for the first time directly demonstrated conformational changes in the environment of the active site upon Ca(2+) binding. Interestingly, this Ca(2+)-induced conformational change can be noted even in the presence of an inhibitor. Forming a complex with sTF further stabilized this conformational change, leading to a more inaccessible active-site located probe.