Uno Carlsson

Comparison of Electron Paramagnetic Resonance Methods to Determine Distances between Spin Labels on Human Carbonic Anhydrase II

Four doubly spin-labeled variants of human carbonic anhydrase II and corresponding singly labeled variants were prepared by site-directed spin labeling. The distances between the spin labels were obtained from continuous-wave electron paramagnetic resonance spectra by analysis of the relative intensity of the half-field transition, Fourier deconvolution of line-shape broadening, and computer simulation of line-shape changes. Distances also were determined by four-pulse double electron-electron resonance. For each variant, at least two methods were applicable and reasonable agreement between methods was obtained. Distances ranged from 7 to 24 A. The doubly spin-labeled samples contained some singly labeled protein due to incomplete labeling. The sensitivity of each of the distance determination methods to the non-interacting component was compared.

The importance of being knotted: effects of the C-terminal knot structure on enzymatic and mechanical properties of bovine carbonic anhydrase II1

In order to better understand the contribution of the knotted folding pattern to the enzymatic and mechanical properties of carbonic anhydrases, we replaced Gln‐253 of bovine carbonic anhydrase II with Cys, which allowed us to measure the mechanical strength of the protein against tensile deformation by avoiding knot tightening. The expressed protein, to our surprise, turned out to contain two conformational isomers, one capable of binding an enzymatic inhibitor and the other not, which led to their separation through affinity chromatography. In near‐ and far‐UV circular dichroism and fluorescence spectra, the separated conformers were very similar to each other and to the wild‐type enzyme, indicating that they both had native‐like conformations. We describe new evidence which supports the notion that the difference between the two conformers is likely to be related to the completeness of the C‐terminal knot formation.

Adsorption to silica nanoparticles of human carbonic anhydrase II and truncated forms induce a molten‐globule‐like structure

Human carbonic anhydrase II pseudo‐wild type (HCAIIpwt) and two truncated variants were adsorbed to ≈9 nm silica nanoparticles. Ellipsometry was used as an indirect measure of protein adsorption. The structural changes of adsorbed proteins were investigated with the use of circular dichroism (CD), intrinsic fluorescence, ANS binding ability and inhibitor binding capacity. It was found that the variants that were truncated at positions 5 and 17 in the N‐terminal end attain a molten‐globule‐like state after interaction with the silica nanoparticles. In contrast, the more stable HCAIIpwt retained most of its native structure after 24 h adsorption to silica nanoparticles. The result suggests that surface induced unfolding may give rise to intermediates similar to those for unfolding induced by, for example GuHCl. Thus, the intermediate observed has some features of the molten globule.

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.

Structural mapping of an aggregation nucleation site in a molten-globule intermediate

Protein aggregation plays an important role in biotechnology and also causes numerous diseases. Human carbonic anhydrase II is a suitable model protein for studying the mechanism of aggregation. We found that a molten globule state of the enzyme formed aggregates. The intermolecular interactions involved in aggregate formation were localized in a direct way by measuring excimer formation between each of 20 site-specific pyrene-labeled cysteine mutants. The contact area of the aggregated protein was very specific, and all sites included in the intermolecular interactions were located in the large β-sheet of the protein, within a limited region between the central β-strands 4 and 7. This substructure is very hydrophobic, which underlines the importance of hydrophobic interactions between specific β-sheet containing regions in aggregate formation.

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.

Denaturation and reactivation of human carbonic anhydrases in guanidine hydrochloride and urea

Abstract 1. 1.Human carbonic anhydrases B and C denatured in concentrated solutions of guanidine hydrochloride have been reactivated to products having 95% and 90%, respectively, of the specific activities of the native enzymes. Conditions are also described for the reactivation of the urea-denatured human B enzyme to yield a product with 85% of the specific activity of the native enzyme. The circular dichroism spectra of the reactivated enzymes closely resemble those of the native enzymes. 2. 2.The kinetics of denaturation of the B and C enzymes in intermediate concentrations of guanidine hydrochloride is complex, and the final products are not readily reactivated. These observations indicate that incorrectly folded molecules, rather than intermediates between the native and the randomly coiled states, are formed under these conditions. 3. 3.The kinetics of the reactivation of the fully denatured enzymes also indicates that “incorrectly” folded molecules are formed. At protein concentrations greater than 0.025 mg/ml, intermolecular reactions markedly interfere with rapid reactivation. Evidence is presented suggesting that disulfide-bridged dimers are not formed to a significant extent in these intermolecular reactions.

High-Resolution Probing of Local Conformational Changes in Proteins by the Use of Multiple Labeling: Unfolding and Self-Assembly of Human Carbonic Anhydrase II Monitored by Spin, Fluorescent, and Chemical Reactivity Probes

Two different spin labels, N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide (IPSL) and (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate (MTSSL), and two different fluorescent labels 5-((((2-iodoacetyl)amino)-ethyl)amino)naphtalene-1-sulfonic acid (IAEDANS) and 6-bromoacetyl-2-dimetylaminonaphtalene (BADAN), were attached to the introduced C79 in human carbonic anhydrase (HCA II) to probe local structural changes upon unfolding and aggregation. HCA II unfolds in a multi-step manner with an intermediate state populated between the native and unfolded states. The spin label IPSL and the fluorescent label IAEDANS reported on a substantial change in mobility and polarity at both unfolding transitions at a distance of 7.4-11.2 A from the backbone of position 79. The shorter and less flexible labels BADAN and MTSSL revealed less pronounced spectroscopic changes in the native-to-intermediate transition, 6.6-9.0 A from the backbone. At intermediate guanidine (Gu)-HCl concentrations the occurrence of soluble but irreversibly aggregated oligomeric protein was identified from refolding experiments. At approximately 1 M Gu-HCl the aggregation was found to be essentially complete. The size and structure of the aggregates could be varied by changing the protein concentration. EPR measurements and line-shape simulations together with fluorescence lifetime and anisotropy measurements provided a picture of the self-assembled protein as a disordered protein structure with a representation of both compact as well as dynamic and polar environments at the site of the molecular labels. This suggests that a partially folded intermediate of HCA II self-assembles by both local unfolding and intermolecular docking of the intermediates vicinal to position 79. The aggregates were determined to be 40-90 A in diameter depending on the experimental conditions and spectroscopic technique used.

Folding around the C-terminus of human carbonic anhydrase II Kinetic characterization by use of a chemically reactive SH-group introduced by protein engineering

We are characterizing the process of refolding of the enzyme human carbonic anhydrase II from the denatured state in guanidine hydrochloride. To describe the folding in defined parts of the protein we use protein engineering to introduce cysteine residues as unique chemically reactive probes. The accessibility of the cysteine SH‐group to the alkylating reagent iodoacetate, at different stages during refolding, is used to give a kinetic description of the folding process. The structuration of the C‐terminal part of the polypeptide chain, which is involved in a unique ‘knot’ topology, was investigated. Our results show that the structure around the C‐terminal, composed of the outermost β‐strands in a dominating β‐structure that extends through the entire protein, is formed relatively late during refolding. In contrast, it was found that β‐strands located in the interior of the protein were structured very rapidly. The final native structure is formed in a process that is slower than those observed for formation of β‐structure.

Folding of β-sheet proteins

Abstract In the past year, interesting new information concerning various aspects of the folding process of β-sheet proteins has been gleaned. Kinetic and equilibrium folding intermediates have been characterized. Studies of extensively denatured states and of model peptide fragments have enabled important steps to be taken towards an understanding of the initiation of the folding process of β-sheet proteins. Site-directed mutagenesis has been used in combination with various probes to monitor folding events.