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We have analysed hydrogen exchange at amide groups to characterise the energy landscape of the 72 amino acid residue protein MerP. From the guanidine hydrochloride (GuHCl) dependence of exchange in the pre-transitional region we have determined free energy values of exchange (DeltaG(HX)) and corresponding m-values for individual amide protons. Detailed analysis of the exchange patterns indicates that for one set of amide protons there is a weak dependence on denaturant, indicating that the exchange is dominated by local fluctuations. For another set of amide protons a linear, but much stronger, denaturant dependence is observed. Notably, the plots of free energy of exchange versus [GuHCl] for 16 amide protons show pronounced upward curvature, and a close inspection of the structure shows that these residues form a well-defined core in the protein. The hydrogen exchange that was measured at various concentrations of NaCl shows an apparent selective stabilisation of this core. Detailed analysis of this exchange pattern indicates that it may originate from selective destabilisation of the unfolded state by guanidinium ions and/or selective stabilisation of the core in the native state by chloride ions.

The binding of protein to solid surfaces often induces changes in the structure, and to investigate these matters we have selected two different protein-nanoparticle systems. The first system concerns the enzyme human carbonic anhydrase II which binds essentially irreversibly to the nanoparticles, and the second system concerns human carbonic anhydrase I which alternate between the adsorbed and free state upon interaction with nanoparticles. Application of the TROSY pulse sequence has allowed high-resolution NMR analysis for both of the protein-nanoparticle systems. For HCAII it was possible to observe spectra of protein when bound to the nanoparticles. The results indicated that HCAII undergoes large rearrangements, forming an ensemble of molten globule-like structures on the surface. The spectra from the HCAI-nanoparticle system are dominated by HCAI molecules in solution. A comparative analysis of variations in intensity from 97 amide resonances in a 1H-15N TROSY spectrum revealed the effects from interaction with nanoparticle on the protein structure at amino acid resolution.

A general problem when analysing NMR spectra that reflect variations in the environment of target molecules is that different resonances are affected to various extents. Often a few resonances that display the largest frequency changes are selected as probes to reflect the examined variation, especially in the case, where the NMR spectra contain numerous resonances. Such a selection is dependent on more or less intuitive judgements and relying on the observed spectral variation being primarily caused by changes in the NMR sample. Second, recording changes observed for a few (albeit significant) resonances is inevitably accompanied by not using all available information in the analysis. Likewise, the commonly used chemical shift mapping (CSM) [Biochemistry 39 (2000) 26, Biochemistry 39 (2000) 12595] constitutes a loss of information since the total variation in the data is not retained in the projection into this single variable. Here, we describe a method for subjecting 2D NMR time-domain data to multivariate analysis and illustrate it with an analysis of multiple NMR experiments recorded at various folding conditions for the protein MerP. The calculated principal components provide an unbiased model of variations in the NMR spectra and they can consequently be processed as NMR data, and all the changes as reflected in the principal components are thereby made available for visual inspection in one single NMR spectrum. This approach is much less laborious than consideration of large numbers of individual spectra, and it greatly increases the interpretative power of the analysis.

We have analyzed the adsorption of protein to the surfaces of silica nanoparticles with diameters of 6, 9, and 15 nm. The effects upon adsorption on variants of human carbonic anhydrase with differing conformational stabilities have been monitored using methods that give complementary information, i.e., circular dichroism (CD), nuclear magnetic resonance (NMR), analytical ultracentrifugation (AUC), and gel permeation chromatography. Human carbonic anhydrase I (HCAI), which is the most stable of the protein variants, establishes a dynamic equilibrium between bound and unbound protein following mixture with silica particles. Gel permeation and AUC experiments indicate that the residence time of HCAI is on the order of approximately 10 min and slowly increases with time, which allows us to study the effects of the interaction with the solid surface on the protein structure in more detail than would be possible for a process with faster kinetics. The effects on the protein conformation from the interaction have been characterized using CD and NMR measurements. This study shows that differences in particle curvature strongly influence the amount of the protein's secondary structure that is perturbed. Particles with a longer diameter allow formation of larger particle-protein interaction surfaces and cause larger perturbations of the protein's secondary structure upon interaction. In contrast, the effects on the tertiary structure seem to be independent of the particles' curvature.

We have analysed the folding energy landscape of the 72 amino acid protein MerP by monitoring native state hydrogen exchange as a function of temperature in the range of 7-55 degrees C. The temperature dependence of the hydrogen exchange has allowed us to determine DeltaG, DeltaH and DeltaC(p) values for the conformational processes that permit hydrogen exchange. When studied with the traditional probes, fluorescence and CD, MerP appears to behave as a typical two-state protein, but the results from the hydrogen exchange analysis reveal a much more complex energy landscape. Analysis at the individual amino acid level show that exchange is allowed from an ensemble of partially unfolded structures (i.e. intermediates) in which the stabilities at the amino acid level form a broad distribution throughout the protein. The formation of partially unfolded structures might contribute to the unusually slow folding of MerP.

The hydration of nonnative states is central to protein folding and stability but has been probed mainly by indirect methods. Here we use water 17O relaxation dispersion to monitor directly the internal and external hydration of α-lactalbumin, lysozyme, ribonuclease A, apomyoglobin and carbonic anhydrase in native and nonnative states. The results show that nonnative proteins are more structured and less solvent exposed than commonly believed. Molten globule proteins preserve most of the native internal hydration sites and have native-like surface hydration. Proteins denatured by guanidinium chloride are not fully solvent exposed but contain strongly perturbed occluded water. These findings shed new light on hydrophobic stabilization of proteins. [ABSTRACT FROM AUTHOR]

The gene merP, coding for a mercuric-ion-binding periplasmic protein (P protein), was cloned into the expression vector pCA. In an Escherichia coli strain bearing the resulting plasmid, the P protein constitutes about 20% of total soluble protein. P protein was purified using ammonium sulfate precipitation and two chromatography steps. Typical yields were 20–30 mg from 7.5 I bacterial culture. The protein is a monomer with a molecular mass of 7500 Da. The periplasmic signal peptide was processed identically in both the recombinant and the wild-type proteins. CD spectra of both proteins were identical and indicated that the structure is highly ordered, containing approximately 80% α-helix. Purification in the presence of excess cysteine resulted in a form of the protein containing two reduced thiols, in agreement with the published sequence which has two cysteine residues. When cysteine was omitted from the purification buffers, no reduced thiol groups could be detected suggesting that the cysteine residues are oxidized. Both of these forms of the protein were found to bind approximately five Hg2+ ions/protein molecule in an apparently non-specific manner. However, in the presence of external thiol compounds, the protein with reduced thiols bound only one Hg2+ ion/protein molecule with an apparent Kd of 3.7 ± 1.3 μM. Under these conditions, the protein with oxidized thiols did not bind Hg2+. The possible physiological role of this protein in Hg2+ detoxification is discussed. [ABSTRACT FROM AUTHOR]

1. The single - SH groups in the human carbonic anhydrases B and C have been modified under denaturing conditions. The modified enzymes recover catalytic activity after dilution of the denaturing medium with buffer. By this method a spin label and a fluorescent probe were specifically introduced into the molecules. 2. The modified and reactivated enzymes have similar kinetic properties, inhibitor-binding constants, circular dichroism spectra, and stabilities towards guanidine hydrochlodde as the native enzymes. However, the esterase activity of the modified C enzyme is reduced to about 50 %. 3. The spectra associated with the probes are insensitive to inhibitor binding in case of the B enzyme, whereas changes of electron paramagnetic resonance spectrum and fluorescence intensity respectively, were observed for the probe-containing C enzymes. The cysteines are located in different parts of the tertiary structures of the homologous B and C enzymes, and these observations suggest that small conformational changes accompanying inhibitor binding are localized to regions of the molecules near the active-site cavity. 4. Dudng denaturation of the spin-labeled B enzyme in 1.7 M guanidine hydrochlodde a transient mobilization of the probe occurs, but the mobility is ultimately reduced to a low level. This observation supports previous evidence that denaturation under these conditions, in or near the transition region, mainly yields incorrectly folded molecules rather than stable intermediates between native and fully denatured molecules. 5. During refolding of fully denatured, spin-labeled B and C enzymes the mobility of the probe is drastically reduced within less than 0.1 s after dilution. This would reflect a very short lifetime of the randomly coiled state under these conditions. [ABSTRACT FROM AUTHOR]