Malin Persson

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.

Heavy Meromyosin Molecules Extending More Than 50 nm above Adsorbing Electronegative Surfaces

In the in vitro motility assay, actin filaments are propelled by surface-adsorbed myosin motors, or rather, myosin motor fragments such as heavy meromyosin (HMM). Recently, efforts have been made to develop actomyosin powered nanodevices on the basis of this assay but such developments are hampered by limited understanding of the HMM adsorption geometry. Therefore, we here investigate the HMM adsorption geometries on trimethylchlorosilane- [TMCS-] derivatized hydrophobic surfaces and on hydrophilic negatively charged surfaces (SiO(2)). The TMCS surface is of great relevance in fundamental studies of actomyosin and both surface substrates are important for the development of motor powered nanodevices. Whereas both the TMCS and SiO(2) surfaces were nearly saturated with HMM (incubation at 120 microg mL(-1)) there was little actin binding on SiO(2) in the absence of ATP and no filament sliding in the presence of ATP. This contrasts with excellent actin-binding and motility on TMCS. Quartz crystal microbalance with dissipation (QCM-D) studies demonstrate a HMM layer with substantial protein mass up to 40 nm above the TMCS surface, considerably more than observed for myosin subfragment 1 (S1; 6 nm). Together with the excellent actin transportation on TMCS, this strongly suggests that HMM adsorbs to TMCS mainly via its most C-terminal tail part. Consistent with this idea, fluorescence interference contrast (FLIC) microscopy showed that actin filaments are held by HMM 38 +/- 2 nm above the TMCS-surface with the catalytic site, on average, 20-30 nm above the surface. Viewed in a context with FLIC, QCM-D and TIRF results, the lack of actin motility and the limited actin binding on SiO(2) shows that HMM adsorbs largely via the actin-binding region on this surface with the C-terminal coiled-coil tails extending >50 nm into solution. The results and new insights from this study are of value, not only for the development of motor powered nanodevices but also for the interpretation of fundamental biophysical studies of actomyosin function and for the understanding of surface-protein interactions in general.

Protein substrate binding induces conformational changes in the chaperonin GroEL. A suggested mechanism for unfoldase activity.

Chaperonins are molecules that assist proteins during folding and protect them from irreversible aggregation. We studied the chaperonin GroEL and its interaction with the enzyme human carbonic anhydrase II (HCA II), which induces unfolding of the enzyme. We focused on conformational changes that occur in GroEL during formation of the GroEL-HCA II complex. We measured the rate of GroEL cysteine reactivity toward iodo[2-14C]acetic acid and found that the cysteines become more accessible during binding of a cysteine free mutant of HCA II. Spin labeling of GroEL withN-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide revealed that this additional binding occurred because buried cysteine residues become accessible during HCA II binding. In addition, a GroEL variant labeled with 6-iodoacetamidofluorescein exhibited decreased fluorescence anisotropy upon HCA II binding, which resembles the effect of GroES/ATP binding. Furthermore, by producing cysteine-modified GroEL with the spin labelN-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide and the fluorescent label 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid, we detected increases in spin-label mobility and fluorescence intensity in GroEL upon HCA II binding. Together, these results show that conformational changes occur in the chaperonin as a consequence of protein substrate binding. Together with previous results on the unfoldase activity of GroEL, we suggest that the chaperonin opens up as the substrate protein binds. This opening mechanism may induce stretching of the protein, which would account for reported unfoldase activity of GroEL and might explain how GroEL can actively chaperone proteins larger than HCA II.

GroEL reversibly binds to, and causes rapid inactivation of, human carbonic anhydrase II at high temperatures.

The initial yield of reactivation of GuHCl denatured human carbonic anhydrase II does not change with temperature between 3 and 35 degrees C. At temperatures above 35 degrees C, the enzymatic activity is not stable, but decreases over time. If the bacterial chaperonin GroEL is present during reactivation, the initial yield is lower compared to the spontaneous reaction at temperatures of 35-50 degrees C. However, unlike the spontaneous reactivation, the enzymatic activity with time in the presence of GroEL. In the presence of GroEL, native HCA II incubated at elevated temperatures will rapidly loose enzymatic activity to the same value as during reactivation at that particular temperature; most of the activity will recover if the temperature is lowered when GroEL is present. It is evident that there is an equilibrium between an inactive intermediate of HCA II, probably bound to GroEL, and active enzyme. Furthermore, proline isomerization is part of the rate-limiting step of refolding even in the presence of GroEL, and it is very noteworthy that prolyl isomerase will influence the refolding of HCA II in the presence of GroEL.

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.

Chiral dendritic polymers for photonic applications

Abstract We present chiral dendrons of different generations accomplished by reacting the hydroxyl groups at the chain ends with (−)menthoxyacetic acid. Subsequent deprotection of the carboxylic acid rendered acid functional chiral dendrons. The acid-functionalized chiral dendrons were doped with divalent cations Cu 2+ , Fe 2+ and Zn 2+ , and trivalent lanthanide cations Nd 3+ and Pr 3+ . We present results on their optical rotatory power along with circular dichroism spectroscopy and results of paramagnetic resonance. The chiral dendrons were shown to influence the electronic transitions of the metal ions (CD spectra). Attempts to characterize the circularly polarized luminescence of the Nd-dendrimer failed due to low quantum yield. The luminescence efficiency was found to be at least one order of magnitude lower than that of a fluorinated and non-chiral dendrimer structure of similar size and coordination structure.

GroEL provides a folding pathway with lower apparent activation energy compared to spontaneous refolding of human carbonic anhydrase II

The kinetics of the refolding of the enzyme, human carbonic anhydrase II (HCA II), at different temperatures, together with the Escherichia coli chaperonin GroEL, has been studied. The Arrhenius plots for the spontaneous, GroEL‐assisted, and GroEL/ES‐assisted refolding of HCA II show that the apparent activation energy (E a) is lower in the presence of the chaperonin GroEL alone than for the spontaneous reaction, whereas the apparent activation energy for the GroEL/ES‐assisted reaction is almost the same as for the spontaneous reaction (85, 46, and 72 kJ/mol, for the spontaneous, GroEL, and GroEL/ES‐assisted reactions, respectively).

Actin Filament Motility Induced Variation of Resonance Frequency and Rigidity of Polymer Surfaces Studied by Quartz Crystal Microbalance

This contribution reports on the quantification of the parameters of the motility assays for actomyosin system using a quartz crystal microbalance (QCM). In particular, we report on the difference in the observed resonance frequency and dissipation of a quartz crystal when actin filaments are stationary as opposed to when they are motile. The changes in QCM measurements were studied for various polymer-coated surfaces functionalized with heavy meromyosin (HMM). The results of the QCM experiments show that the HMM-induced sliding velocity of actin filaments is modulated by a combination of the viscoelastic properties of the polymer layer including the HMM motors.

Surface-Controlled Properties of Myosin Studied by Electric Field Modulation

The efficiency of dynamic nanodevices using surface-immobilized protein molecular motors, which have been proposed for diagnostics, drug discovery, and biocomputation, critically depends on the ability to precisely control the motion of motor-propelled, individual cytoskeletal filaments transporting cargo to designated locations. The efficiency of these devices also critically depends on the proper function of the propelling motors, which is controlled by their interaction with the surfaces they are immobilized on. Here we use a microfluidic device to study how the motion of the motile elements, i.e., actin filaments propelled by heavy mero-myosin (HMM) motor fragments immobilized on various surfaces, is altered by the application of electrical loads generated by an external electric field with strengths ranging from 0 to 8 kVm(-1). Because the motility is intimately linked to the function of surface-immobilized motors, the study also showed how the adsorption properties of HMM on various surfaces, such as nitrocellulose (NC), trimethylclorosilane (TMCS), poly(methyl methacrylate) (PMMA), poly(tert-butyl methacrylate) (PtBMA), and poly(butyl methacrylate) (PBMA), can be characterized using an external field. It was found that at an electric field of 5 kVm(-1) the force exerted on the filaments is sufficient to overcome the frictionlike resistive force of the inactive motors. It was also found that the effect of assisting electric fields on the relative increase in the sliding velocity was markedly higher for the TMCS-derivatized surface than for all other polymer-based surfaces. An explanation of this behavior, based on the molecular rigidity of the TMCS-on-glass surfaces as opposed to the flexibility of the polymer-based ones, is considered. To this end, the proposed microfluidic device could be used to select appropriate surfaces for future lab-on-a-chip applications as illustrated here for the almost ideal TMCS surface. Furthermore, the proposed methodology can be used to gain fundamental insights into the functioning of protein molecular motors, such as the force exerted by the motors under different operational conditions.