Lars-Göran Mårtensson

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.

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.

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.

Role of an evolutionarily invariant serine for the stability of human carbonic anhydrase II

There are several evolutionarily invariant amino acids in the primary structures of all known isoenzymes of carbonic anhydrase. One of these is Ser-29 which is situated in the peripheral part of the active site interacting by hydrogen bonds with amino acids located nearby in the tertiary structure. Furthermore, the neighbourhood of Ser-29, composed of Gln-28, Pro-30, Tyr-194, Ser-197 and Trp-209, has a totally invariant structure. The structural role of Ser-29 was investigated by site-directed mutagenesis. The stability of two enzyme mutants, where Ser-29 was replaced by alanine and cysteine, towards denaturation by guanidine-HCl was studied. Changing Ser-29 to Ala resulted in a destabilization by 2.6 kcal/mol, corresponding to the loss of 2-3 hydrogen bonds. Interestingly, Ser-29 is within hydrogen bond distance to Tyr-194, Ser-197 and Trp-209 in the tertiary structure. Therefore, rupture of these interactions caused by the Ser-29----Ala substitution could explain the observed destabilization of this enzyme variant. Substituting cysteine for Ser-29 gives rise to a drastic decrease in the stability of the protein (change in midpoint concentration of denaturation from 0.96 M to less than 0.1 M guanidine-HCl) despite the minor structural change (O----S atom). This destabilization corresponds to approx. 7-8 kcal/mol and cannot be explained by changes in hydrogen bond pattern only, but must also include unfavourable conformational changes to avoid van der Waals collisions originating from the somewhat larger thiol group.

Dramatic Stabilization of the Native State of Human Carbonic Anhydrase II by an Engineered Disulfide Bond

To find a disulfide pair that could stabilize the enzyme human carbonic anhydrase II (HCA II), we grafted the disulfide bridge from the related and unusually stable carbonic anhydrase form from Neisseria gonorrhoeae (NGCA) into the human enzyme. Thus, the two Cys residues at positions 23 and 203 were engineered into a pseudo-wild-type form of HCA II (C206S), giving the mutant C206S/A23C/L203C. The disulfide bond was not formed spontaneously. The native state of the reduced form of the mutant was markedly destabilized (2.9 kcal/mol) compared to that of HCA II. Formation of a disulfide bridge was achieved by treatment by oxidized glutathione. This led to a significant stabilization of the native conformation. Compared to HCA II the unfolding midpoint for the variant was increased from 0.9 to 1.7 M guanidine HCl, corresponding to a stabilization of 3.7 kcal/mol. This makes the human enzyme almost as stable as the model protein NGCA, for which the unfolding of the native state has a midpoint at 2.1 M guanidine HCl. The stabilized protein underwent, contrary to all other investigated variants of HCA II, an apparent two-state unfolding transition, as judged from intrinsic Trp fluorescence measurements. A molten-globule intermediate is nevertheless formed but is suppressed because of the high denaturant pressure it faces upon rupture of the native state.

Characterization of a novel sequence variant, TPMT*28, in the human thiopurine methyltransferase gene

Background The activity of the human enzyme thiopurine methyltransferase (TPMT) varies greatly between individuals because of genetic polymorphism. TPMT is involved in the detoxification and activation of thiopurines such as 6-mercaptopurine, 6-thioguanine, and azathioprine. These drugs are used in the treatment of acute lymphoblastic leukemia and inflammatory bowel disease. A total of 29 sequence variants have been identified so far in the TPMT gene. However, most of these variants are rare and not fully characterized. Methods and results In this study, we describe the identification and characterization of a novel TPMT sequence variant, originally found in a Swedish man of Italian origin. Sequencing of the variable number tandem repeats region of the TPMT promoter and exons III–X revealed a T-to-C transition at nucleotide 611, causing an amino acid substitution from isoleucine to threonine at amino acid 204, positioned in an α-helix, approximately 16 Å from the active site. This new variant was found in the patient and in his son. Both had intermediate enzyme activity (8.1 U/ml packed red blood cells and 8.8 U/ml packed red blood cells, respectively) and neither carried other variants in the coding region of the gene. To be able to study this variant in more detail, the TPMT*28 variant was expressed in Escherichia coli, and an in-vitro characterization of the variant revealed that the protein was destabilized and showed a stronger tendency towards degradation at 37°C than the wild-type protein. The individuals carrying the TPMT*28 variant had less TPMT protein and lower TPMT activity in both red and white blood cells compared with a wild-type control. Conclusions We present a detailed in-vivo and in-vitro characterization of a novel TPMT sequence variant (TPMT*28) causing decreased TPMT activity. Individuals carrying TPMT*28 might have an increased risk for developing severe side effects if treated with conventional doses of thiopurines.

Phase memory relaxation times of spin labels in human carbonic anhydrase II: pulsed EPR to determine spin label location.

Phase memory relaxation times (T(M) or T(2)) of spin labels in human carbonic anhydrase II (HCA II) are reported. Spin labels (N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide, IPSL) were introduced at cysteines, by site-directed mutagenesis at seven different positions in the protein. By two pulse electron paramagnetic resonance (EPR), electron spin echo decays at 45 K are measured and fitted by stretched exponentials, resulting in relaxation parameters T(M) and x. T(M) values of seven positions are between 1.6 micros for the most buried residue (L79C) and 4.7 micros for a residue at the protein surface (W245C). In deuteriated buffer, longer T(M) are found for all but the most buried residues (L79C and W97C), and electron spin echo envelop modulation (ESEEM) of deuterium nuclei is observed. Different deuterium ESEEM patterns for W95C and W16C (surface residue) indicate differences in the local water concentration, or accessibility, of the spin label by deuterium. We propose T(M) as a parameter to determine the spin label location in proteins. Furthermore, these systems are interesting for studying the pertaining relaxation mechanism.

Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance

The self‐assembling MexA‐MexB‐OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR‐wt as well as a selected set of MDR single mutants distant from the proposed DNA‐binding helix. Although DNA affinity and MexA‐MexB‐OprM repression were both drastically impaired in the selected MexR‐MDR mutants, MexR‐wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR‐MDR mutants, secondary structure content and oligomerization properties were very similar to MexR‐wt despite their lack of DNA binding. Despite this, the MexR‐MDR mutants showed highly varying stabilities compared with MexR‐wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA‐binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR‐wt in both free and DNA‐bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations—stability, domain interactions, and internal hydrophobic surfaces—are also critical for the regulation of MexR DNA binding.