Limitations of the linear and the projection approximations in three-dimensional transmission electron microscopy of fully hydrated proteins.

We establish expressions for the linear and quadratic terms in the series expansion of the phase and the phase and amplitude object description of imaging thin specimens by transmission electron microscopy. Based on these expressions we simulate the corresponding contributions to images of unstained protein complexes of varying thickness and arrive at an estimate for how much each term contributes to the contrast of the image. From this we can estimate a maximum specimen thickness for which the weak phase and the weak amplitude and phase object approximation (and therefore linear imaging) is still reasonably accurate. When discussing thick specimens it is also necessary to consider limitations due to describing the image as a filtered projection of the specimen, since the different layers of the specimen are not imaged with the same defocus value. We therefore compared simulations based on the projection approximation with the more accurate multislice model of image formation. However, we find that the errors due to nonlinear image contributions are greater than those due to the defocus gradient for the defocus values chosen for the simulations. Finally, we study how the discussed nonlinear image contributions and the defocus gradient affect the quality of three-dimensional reconstructions. We find that three-dimensional reconstructions reach high resolution when at the same time exhibiting localized systematic structural errors.

Ion pairs and the thermotolerance of proteins from hyperthermophiles: a "traffic rule" for hot roads.

The proteins from hyperthermophilic organisms maintain their biologically active structure at temperatures that are significantly higher than the denaturation temperatures of their mesophilic counterparts. The fact that there is usually a high degree of sequence and structural homology between these two classes of proteins suggests that the source of this extreme thermal tolerance is hidden in the delicate balance of the non-covalent interactions. Among the large number of factors identified in the literature as being responsible for the thermostability of these proteins, this article focuses on electrostatic interactions. It demonstrates that the optimization of electrostatic interactions by increasing of the number of salt bridges is a driving force for enhancement of the thermotolerance of proteins from hyperthermophilic microorganisms. This feature is less evident in proteins from thermophilic organisms and is absent from mesophile-derived proteins.

Electrostatic properties of two porin channels from Escherichia coli.

The electrostatic interactions in the channels of OmpF and PhoE porins from Escherichia coli were analysed on the basis of a macroscopic multi-dielectric model of the protein-membrane complex derived from the respective porin X-ray structures. The membrane was represented as layers of distinct dielectric constants corresponding to the aliphatic core and the polar head groups of the lipids. The pKa values of the titratable groups and the electrostatic field in the region of the channel were calculated by the finite difference technique. In spite of the differences in sequences and charge constellations, the calculated electrostatic properties of the two porins are similar in several aspects: (1) unusual titration behaviour (pKa below 7) was found for some groups of the cluster of basic residues at the constriction of the pore; (2) a number of acidic groups buried between the internal loop and the barrel wall are stabilized in their protonated forms at neutral pH; (3) there is a strong transverse electrostatic field in the channel characterized by a screw-like form. The strength of the field is greatest at the region of the constriction zone. This would facilitate the diffusion of solutes with a large dipole moment such as free amino acids. Differences between the electrostatic fields of OmpF and PhoE are mainly confined to that end of the pore that faces the cell exterior in vivo. In OmpF the electrostatic potential is close to zero in this region of the channel, whereas a positive potential was found in PhoE. It was shown that the experimentally observed difference in ion selectivity of the two porins can largely be attributed to this distinct electrostatic property.

DNA-binding surface of the Sso7d protein from Sulfolobus solfataricus.

We have used nuclear magnetic resonance (n.m.r.) spectroscopy to identify the DNA-binding surface of the abundant, small and basic protein Sso7d from the hyperthermophilic archaebacterium Sulfolobus solfataricus. The Sso7d protein was previously found to bind strongly to double-stranded DNA sequences and to protect DNA from thermal denaturation, indicating that it might assume a similar function in vivo. Several amide resonances in two-dimensional n.m.r. 1H, 15N correlation spectra of 15N-enriched Sso7d are shifted and broadened upon addition of small amounts of ten base-pair or 19 base-pair duplex DNA oligomers under conditions where Sso7d-DNA complexes exchange rapidly on the n.m.r. time scale. The locations of the corresponding amides in the Sso7d structure define the surface that interacts with DNA. This surface coincides with a continuous region of strong positive electrostatic potential, which was calculated by means of numerical solution of the Poisson-Boltzmann equation. A model of the non-specific Sso7d-DNA complex is suggested based on the present data and previously obtained evidence that Sso7d interacts with the DNA major groove. The protein-DNA interface consists of a triple-stranded beta-sheet, which interacts with the DNA major groove and a reverse turn connecting the two strands of a double-stranded beta-sheet, which interacts with the minor groove. We note that the five (of 14) lysine side-chains that are specifically subjected to N zeta-monomethylation in the cell are located on surfaces of Sso7d that are exposed to the solvent in the proposed Sso7d-DNA complex.

Thermal unfolding of the DNA-binding protein Sso7d from the hyperthermophile Sulfolobus solfataricus.

Thermal unfolding of the small hyperthermophilic DNA-binding protein Sso7d was studied by circular dichroism spectroscopy and differential scanning calorimetry. The unfolding transition can be described by a reversible two state process. Maximum stability was observed in the region between pH 4.5 and 7.0 where Sso7d unfolds with a melting temperature between 370.8 to 371.9 K and an unfolding enthalpy between 62.9 and 65.4 kcal/mol. The heat capacity differences between the native and the heat denatured states obtained by differential scanning calorimetry (620 cal/(molK)) and circular dichroism spectroscopy (580 cal/(mol K)) resulted in comparable values. The thermodynamic reason for the high melting temperature of Sso7d is the shallow stability curve with a broad free energy maximum, corresponding to the relatively small heat capacity change which was obtained. The calculated stability curve shows that Sso7d has, despite of its high melting temperature, an only moderate intrinsic stability, which reaches its maximum (approximately 7 kcal/mol) at 282 K. Sso7d is particularly poorly stabilized (approximately 1 kcal/mol) at the maximum physiological growth temperature of Sulfolobus solfataricus. Sso7d has furthermore untypically low specific enthalpy (0.99 kcal/(mol residue)) and entropy (2.99 cal/(mol K)) values at convergence temperatures. No significant differences in thermal stability of the partially methylated Sso7d from Sulfolobus solfataricus and the cloned non-methylated form of the protein expressed in Escherichia coli were observed.

Optimization of the electrostatic interactions between ionized groups and peptide dipoles in proteins.

The three-dimensional optimization of the electrostatic interactions between the charged amino acid residues and the peptide partial charges was studied by Monte-Carlo simulations on a set of 127 nonhomologous protein structures with known atomic coordinates. It was shown that this type of interaction is very well optimized for all proteins in the data set, which suggests that they are a valuable driving force, at least for the native side-chain conformations. Similar to the optimization of the charge-charge interactions (Spassov VZ, Karshikoff AD, Ladenstein R, 1995, Protein Sci 4:1516-1527), the optimization effect was found more pronounced for enzymes than for proteins without enzymatic function. The asymmetry in the interactions of acidic and basic groups with the peptide dipoles was analyzed and a hypothesis was proposed that the properties of peptide dipoles are a factor contributing to the natural selection of the basic amino acids in the chemical composition of proteins.

The optimization of protein-solvent interactions: thermostability and the role of hydrophobic and electrostatic interactions.

Protein-solvent interactions were analyzed using an optimization parameter based on the ratio of the solvent-accessible area in the native and the unfolded protein structure. The calculations were performed for a set of 183 nonhomologous proteins with known three-dimensional structure available in the Protein Data Bank. The dependence of the total solvent-accessible surface area on the protein molecular mass was analyzed. It was shown that there is no difference between the monomeric and oligomeric proteins with respect to the solvent-accessible area. The results also suggested that for proteins with molecular mass above some critical mass, which is about 28 kDa, a formation of domain structure or subunit aggregation into oligomers is preferred rather than a further enlargement of a single domain structure. An analysis of the optimization of both protein-solvent and charge-charge interactions was performed for 14 proteins from thermophilic organisms. The comparison of the optimization parameters calculated for proteins from thermophiles and mesophiles showed that the former are generally characterized by a high degree of optimization of the hydrophobic interactions or, in cases where the optimization of the hydrophobic interactions is not sufficiently high, by highly optimized charge-charge interactions.

Optimization of the electrostatic interactions in proteins of different functional and folding type.

The 3-dimensional optimization of the electrostatic interactions between the charged amino acid residues was studied by Monte Carlo simulations on an extended representative set of 141 protein structures with known atomic coordinates. The proteins were classified by different functional and structural criteria, and the optimization of the electrostatic interactions was analyzed. The optimization parameters were obtained by comparison of the contribution of charge-charge interactions to the free energy of the native protein structures and for a large number of randomly distributed charge constellations obtained by the Monte Carlo technique. On the basis of the results obtained, one can conclude that the charge-charge interactions are better optimized in the enzymes than in the proteins without enzymatic functions. Proteins that belong to the mixed alpha beta folding type are electrostatically better optimized than pure alpha-helical or beta-strand structures. Proteins that are stabilized by disulfide bonds show a lower degree of electrostatic optimization. The electrostatic interactions in a native protein are effectively optimized by rejection of the conformers that lead to repulsive charge-charge interactions. Particularly, the rejection of the repulsive contacts seems to be a major goal in the protein folding process. The dependence of the optimization parameters on the choice of the potential function was tested. The majority of the potential functions gave practically identical results.

Molecular electroporation: a unifying concept for the description of membrane pore formation by antibacterial peptides, exemplified with NK-lysin.

The antibacterial activity of many small, positively charged peptides and proteins is based on pore formation in lipid bilayers. It is here proposed to arise from an electroporation effect. This hypothesis is supported by calculations of the electrostatic potential of NK-lysin associated to a membrane. For a significant area of the protein-membrane interface, the electrostatic potential is found to be above the minimum threshold for electroporation. A single highly charged alpha-helical segment of NK-lysin is mainly responsible for this effect. It is experimentally demonstrated that a peptide comprising this helix has antibacterial activity. We propose that superficial association to membranes suffices to trigger electroporation, provided the peptide is sufficiently charged. The effect is referred to as molecular electroporation.

Model for calculation of electrostatic interactions in unfolded proteins.

An approach for the calculation of electrostatic interactions and titration properties of unfolded polypeptide chains (denatured proteins) is proposed. It is based on a simple representation of the denatured proteins as a state with titratable sites distributed on the surface of a sphere, radius of which is assumed to be equal to the radius of gyration, R(g), of an unfolded molecule. Distances between the charges, d, obey constraints arising from the protein sequence. Criteria for evaluation of the parameters R(g) and d were obtained from computer simulations on a polypeptide consisting of 20 identical amino acids (polylysine). The model was applied for calculation of titration curves of denatured barnase and staphylococcal nuclease. It was demonstrated that the approach proposed gives considerably better agreement with the experimental data than the commonly used null approximation. It was also found that titration properties of denatured proteins are slightly, but distinguishably influenced by the amino-acid sequence of the protein.

A simple algorithm for the calculation of multiple site titration curves.

A simple algorithm for the calculation of multiple site titration curves is proposed. It is based on a hybridization of two computational techniques: (i) a modified Tanford-Roxby iterative procedure [Tanford and Roxby (1972) Biochemistry, 11, 2193-2198] and (ii) the Boltzmann statistics. The sites characterized by strong electrostatic coupling were selected for statistical mechanical treatment, whereas all other sites were treated by means of the modified Tanford-Roxby procedure. The selection of the two sets was made on the basis of a criterion related to the interaction energy between the titratable sites in the protein molecule. The algorithm was tested for bovine pancreatic trypsin inhibitor and the pK values calculated were discussed in the light of experimental data and theoretical results obtained by other authors. The algorithm can easily be coded and incorporated into any program package for the calculation of electrostatic interactions in proteins.

Proteins from thermophilic and mesophilic organisms essentially do not differ in packing.

The role of the packing density in the elevation of thermal stability of proteins from thermophilic organisms is widely discussed in the literature. In the present study, this issue was reconsidered in the scale of an unbiased set of protein structures. Partial specific volumes, void and cavity volumes were calculated for a set of 80 non-homologous proteins and for 24 proteins from thermophilic organisms and analysed in the context of their possible role in thermal stabilization. The results showed that there is no significant difference between the two sets in respect to the partial specific volume and cavity volume. The proteins from thermophilic organisms showed a slight tendency of increasing void volume, i.e. reducing the packing density. However this observation was not confirmed by the comparison of this parameter for proteins within different structural families. The results suggested that neither the reduction of the packing density nor the reduction of the packing defects can be considered as a common mechanism for increasing the thermal stability of the proteins from thermophilic organisms. Combining the result from this and our previous study we concluded that the electrostatic interactions seem to be a common factor regulating the thermal tolerance of proteins from thermostable organisms.

Electrostatic evidence for the activation of the glutathione thiol by Tyr7 in pi-class glutathione transferases.

A number of spectrophotometric studies [Graminski, G.F., Kubo, Y. & Armstrong, R.N. (1989) Biochemistry 28, 3562-3568; Liu, S., Zhang, P., Ji, X., Johnson, W.W., Gilliland, G.L. & Armstrong, R.N. (1992) J. Biol. Chem. 267, 4296-4299] have recently shown that the glutathione (GSH) thiol is deprotonated when it is in complex with glutathione S-transferase. Different models have been proposed for the activation of the glutathione S gamma, all pointing out the key role of active-site residue Tyr7. It remains unclear, however, how Tyr7 is actually involved in this process. In this paper we present an analysis of the electrostatic potential in the region of the active site of a pi-class GSH transferase. This analysis provides evidence that the titration behaviour of the absorption band of the E.GSH complex with a pK between 6 and 7 [Liu, S., Zhang, P., Ji, X., Johnson, W.W., Gilliland, G.L. & Armstrong, R.N. (1992) J. Biol. Chem. 267, 4296-4299] should rather be explained by the protonation/deprotonation equilibrium of Tyr7 than by the protonation/deprotonation equilibrium of the GSH thiol group itself. On the basis of this conclusion, a mechanism for activation of GSH is proposed: the Tyr7 OH group is deprotonated by the influence of the protein charge constellation and the peptide dipoles. Thus it acts as a general base, promotes proton abstraction from the GSH thiol and creates a thiolate anion with high nucleophilic reactivity.

Ionization properties of titratable groups in ribonuclease T1. II. Electrostatic analysis.

The experimental NMR data for the individual titratable groups in ribonuclease T1 presented in the preceding paper were analysed by means of a continuum dielectric model. The role of two factors, the alteration of hydrogen loci on the ionizable groups and the conformational flexibility, were analysed. It was suggested that the position of the titratable hydrogen is essential mainly for strongly interacting groups. For groups which are accessible to the solvent and whose ionization is not coupled with the ionization of neighbouring groups, this factor can be neglected. The influence of the conformational flexibility on the electrostatic interactions becomes apparent for the environment of K25. For some strongly interacting groups, non-sigmoidal ionization curves were calculated. On this basis the pH dependence of the NMR chemical shift of the 13Cepsilon2 resonance of H27, whose ionization is coupled with E82, was reproduced.

Role of the C-terminal chain in human interferongamma stability: an electrostatic study.

Electrostatic interactions in two structures of human interferon gamma (hIFNgamma), corresponding to interferon molecule alone and bound to its receptor, were analyzed on the basis of a continuum dielectric model. It was found that a number of titratable groups, mainly basic, show large pK shifts and remain in their neutral forms at physiologically relevant pH. The fact that these groups are largely common to both structures and that most of them belong to the set of most conserved sites suggests that this is a property inherent to the hIFNgamma molecule rather than an artifact of the crystal packing. His111 was also found deprotonated at neutral pH. It was concluded that receptor recognition involving His111 is driven by aromatic coupling of His111 and Tyr52 from the receptor rather than by electrostatic interactions. The structure corresponding to hIFNgamma in complex with its receptor shows a reduction in number and in degree of desolvation of the buried titratable sites. This finding suggested that on receptor binding, hIFNgamma adopts energetically more favorable, relaxed, conformation. It was experimentally shown that in contrast to the full-size hIFNgamma, the construct having 21 amino acid residues deleted from the C-terminus is soluble. The hydrophobicity profile analysis suggested that factors other than the exposure of hydrophobic parts of the molecule are responsible for the low stability and propensity for aggregation. On the basis of these results, it was assumed that the electrostatic influence of the C-terminal part contributes particularly to the low solvent exposure of the titratable groups, and hence to the low structural stability and propensity for aggregation of the recombinant hIFNgamma. Proteins 2001;43:125-133.

Preliminary crystallographic analysis of an extremely thermostable glutamate dehydrogenase from the archaeon Pyrococcus woesei.

The extremely heat-stable glutamate dehydrogenase (GluDH) from Pyrococcus woesei was crystallized by the hanging-drop vapour-diffusion method. Crystals suitable for X-ray crystallographic investigations were obtained using polyethylene glycol (PEG) 4000 and ammonium acetate as precipitating agents. The crystals obtained diffract to a resolution of 2.8 A, have a prismatic shape and grow up to 0.5 mm in their maximal dimension. They belong to the triclinic system (space group P1; a = 90.9, b = 92.8, c = 107.1 A, alpha = 69.1, beta = 80.7, Vgamma = 65.0 degrees ) with a unit-cell volume of 765052 A(3) which accommodates one GluDH hexamer of 276 kDa. The averaged density of the crystal determined by Ficoll-gradient centrifugation is 1.15 g cm(-3), which corresponds to a molecular mass of 255 kDa in the unit cell. A native data set has been collected on an MAR image-plate system using Cu Kalpha radiation. The completeness of the data set in the range 316-3 A is 74%, and contains 64% of the data in the outer shell (3.4-2.8 A), with an average R(merge) value of 9%. Calculation of self-rotation functions revealed the 32 symmetry of the hexamer; 3 non-crystallographic twofold axes were found at a distance of 120 degrees in a plane perpendicular to the non-crystallographic threefold axis.

Well ordered crystals of a short-chain alcohol dehydrogenase from Drosophila lebanonensis: re-evaluation of the crystallographic data and rotation-function analysis.

Alcohol dehydrogenase prepared from Drosophila lebanonensis yields well ordered plate-like crystals which diffract to better than 2.3 A resolution. The crystals belong to space group P2(1) of the monoclinic system; the unit-cell dimensions are a = 65.25, b = 55.77, c = 70.02 A, alpha = 90, beta = 107.08, gamma = 90 degrees. The asymmetric unit of the crystal cell is most probably occupied by a dimer, corresponding to a packing density of 2.15 A(3) Da-L. The orientation of the non-crystallographic twofold symmetry axes is determined by analysis of a self-rotation function calculated with native intensity data.

Ionization properties of titratable groups in ribonuclease T1. I. pKa values in the native state determined by two-dimensional heteronuclear NMR spectroscopy.

pKa values of amino acid side chains of ribonuclease T1 have been determined from the pH dependence of 13C and 15N resonances. It was possible to derive pKa values of single protonation or deprotonation sites of carboxylate and imidazole groups. Deviations from pKa values of free amino acids could be interpreted with electrostatic interactions of corresponding side chains with the protein environment. In particular, the interaction between H27 and E82 led to an increase of the H27 pKa and a decrease of the E82 pKa. The pKa of E28 at the C-terminal end of the alpha-helix was increased because of the dipolar character of the alpha-helix. D76 did not titrate in the investigated pH range of about 2-9. From the chemical shift value this buried side chain seems to be protonated. The pKa values of side chains in the active site deviate from a normal behaviour. The lower pKa value of E58 may be interpreted with the close proximity of this side chain with positively charged H40 and R77. A novel two-dimensional 1H(13Cdelta)13Cgamma correlation experiment was developed to observe the pH dependence of the chemical shifts of the Cgamma resonances of histidine residues. From the inspection of the Cgamma chemical shift-pH profiles it was possible to determine the predominant tautomeric form for the histidine residues at higher pH values.

Thermal unfolding and conformational stability of the recombinant domain II of glutamate dehydrogenase from the hyperthermophile Thermotoga maritima.

Domain II (residues 189-338, M(r) = 16 222) of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was used as a model system to study reversible unfolding thermodynamics of this hyperthermostable enzyme. The protein was produced in large quantities in E.COLI: using a T7 expression system. It was shown that the recombinant domain is monomeric in solution and that it comprises secondary structural elements similar to those observed in the crystal structure of the hexameric enzyme. The recombinant domain is thermostable and undergoes reversible and cooperative thermal unfolding in the pH range 5.90-8.00 with melting temperatures between 75.1 and 68.0 degrees C. Thermal unfolding of the protein was studied using differential scanning calorimetry and circular dichroism spectroscopy. Both methods yielded comparable values. The analysis revealed an unfolding enthalpy at 70 degrees C of 70.2 +/- 4.0 kcal/mol and a DeltaC(p) value of 1.4 +/- 0.3 kcal/mol K. Chemical unfolding of the recombinant domain resulted in m values of 3.36 +/- 0.10 kcal/mol M for unfolding in guanidinium chloride and 1.46 +/- 0.04 kcal/mol M in urea. The thermodynamic parameters for thermal and chemical unfolding equilibria indicate that domain II from T.MARITIMA: glutamate dehydrogenase is a thermostable protein with a DeltaG(max) of 3.70 kcal/mol. However, the thermal and chemical stabilities of the domain are lower than those of the hexameric protein, indicating that interdomain interactions must play a significant role in the stabilization of T. MARITIMA: domain II glutamate dehydrogenase.

Thermal unfolding of small proteins with SH3 domain folding pattern.

The thermal unfolding of three SH3 domains of the Tec family of tyrosine kinases was studied by differential scanning calorimetry and CD spectroscopy. The unfolding transition of the three protein domains in the acidic pH region can be described as a reversible two-state process. For all three SH3 domains maximum stability was observed in the pH region 4.5 < pH < 7.0 where these domains unfold at temperatures of 353K (Btk), 342K (Itk), and 344K (Tec). At these temperatures an enthalpy change of 196 kJ/mol, 178 kJ/mol, and 169 kJ/mol was measured for Btk-, Itk-, and Tec-SH3 domains, respectively. The determined changes in heat capacity between the native and the denatured state are in an usual range expected for small proteins. Our analysis revealed that all SH3 domains studied are only weakly stabilized and have free energies of unfolding which do not exceed 12-16 kJ/mol but show quite high melting temperatures. Comparing unfolding free energies measured for eukaryotic SH3 domains with those of the topologically identical Sso7d protein from the hyperthermophile Sulfolobus solfataricus, the increased melting temperature of the thermostable protein is due to a broadening as well as a significant lifting of its stability curve. However, at their physiological temperatures, 310K for mesophilic SH3 domains and 350K for Sso7d, eukaryotic SH3 domains and Sso7d show very similar stabilities.