Per-Ola Freskgård

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.

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.

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.

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.

GroEL/ES-mediated refolding of human carbonic anhydrase II: role of N-terminal helices as recognition motifs for GroEL

The presence of GroEL/ES during the refolding of human carbonic anhydrase II (pseudo-wild type) was found to increase the yield of active enzyme from 65 to 100%. This chaperone action on the enzyme could be obtained by adding GroEL alone, and the time-course in that case was only moderately slower than the spontaneous process. Truncated forms of carbonic anhydrase, in which N-terminal helices were removed, also served as protein substrates for GroEL/ES. This demonstrates that N-terminally located helices are not obligatory as recognition motifs.

Versatile stopped-flow apparatus with adjustable pistons

Abstract A stopped-flow apparatus with many potential applications is described, in which the vertical design makes operaton easier. In addition, the system has adjustable driving pistons that facilitate refilling of syringes and changing of reaction solutions without allowing air to enter the system. This means that the use of reservoir syringes is redundant. The driving pistons can easily be adjusted to withstand different pressures and temperatures and the O-rings are simple to change when worn out. The vertical stopped-flow system is also flexible, as syringes of different size can be used and are easily changed when, for instance, different dilution ratios are needed.