Biomedical subjects

Andrey Karshikoff

Publications and source records attributed to Andrey Karshikoff.

12 recordsLinked to original sources

PubMed2006

Stability and folding/unfolding kinetics of the homotrimeric coiled coil Lpp-56.

The 56 amino acid long protein moiety of the E. coli outer membrane lipoprotein (Lpp-56) contains a 4,3 hydrophobic heptad repeat and forms a parallel, in-register trimeric coiled coil in solution. The unconventional structural properties of Lpp-56 make it an interesting experimental object to study the folding of a trimeric coiled coil. Folding is unusually slow at low temperatures, and the rates of folding and unfolding are balanced in such a way that the thermodynamic equilibrium is established after considerable time at high temperatures or in the presence of denaturants. Here, we examine the stability and the folding/unfolding kinetics of Lpp-56 at neutral pH using GdmCl as the denaturant. Folding and unfolding appear to represent structural transitions between the folded trimer and the unfolded monomer, without detectable intermediates. For the first time, we estimated the unfolding free energy from a direct measurement at equilibrium. Our estimate of DeltaG(U) = 79 +/- 10 kJ mol(-)(1) compares very well with DeltaG(U) approximately 76-88 kJ mol(-)(1) obtained from the kinetic rate constants of refolding (7 x 10(5) M(-)(2) s(-)(2)) and unfolding (10(-)(9) to 10(-)(11) s(-)(1)) and is almost half of the DeltaG(U) recently suggested using a different methodology (Dragan, A. I., Potekhin, S., Sivolob, A., Lu, M., and Privalov, P. L. (2004) Biochemistry 43, 14891-14900). Because GdmCl attenuates electrostatic interactions, the discrepancy can be partly explained by an electrostatic component of the unfolding barrier. The combined information illustrates the difficulties in obtaining a precise biophysical description of proteins that exhibit unusual kinetic properties. Lpp-56 is the first coiled coil for which a high unfolding kinetic barrier has been experimentally demonstrated.

Bacterial Outer Membrane Proteins↗

PubMed2004

Charge sequence coding in statistical modeling of unfolded proteins.

Unfolded proteins recently attracted attention due to accumulation of experimental evidences for their significant role in different life processes. Modeling of electrostatic interactions (EI) in unfolded state of proteins is becoming increasingly important as well. In this paper, we stress on the importance of how the sequence of charged residues of a given protein is incorporated into the models for calculation of EI in the unfolded state. On the basis of the distributions of distances between titratable sites of charged residues calculated for polypeptide chains of various compositions, it was found that the distance distribution for a pair of residues, located close to each other along the sequence of a protein, depends on what residues constitute the pair in question. It was concluded that the consideration of these residue-specific distributions is essential for a statistical model to be accurate from the physical point of view. It was suggested that use of distance intervals in the spherical model of unfolded proteins accounts better for the charge sequence than the set of single distance values. This was illustrated by comparison of the pK values of the titratable groups of the unfolded N-terminal SH3 domain of the Drosophila protein drk to the available experimental data.

Amino Acids↗

PubMed2004

The 2.9A resolution crystal structure of malate dehydrogenase from Archaeoglobus fulgidus: mechanisms of oligomerisation and thermal stabilisation.

The crystal structure of malate dehydrogenase from the hyperthermophilic archaeon Archeoglobus fulgidus, in complex with its cofactor NAD, was solved at 2.9A resolution. The crystal structure shows a compact homodimer with one coenzyme bound per subunit. The substrate binding site is occupied by a sulphate ion. In order to gain insight into adaptation mechanisms, which allow the protein to be stable and active at high temperatures, the 3D structure was compared to those of several thermostable and hyperthermostable homologues, and to halophilic malate dehydrogenase. The hyperthermostable A. fulgidus MalDH protein displays a reduction of the solvent-exposed surface, an optimised compact hydrophobic core, a high number of hydrogen bonds, and includes a large number of ion pairs at the protein surface. These features occur concomitantly with a reduced number of residues in the protein subunit, due to several deletions in loop regions. The loops are further stiffened by ion pair links with secondary structure elements. A. fulgidus malate dehydrogenase is the only dimeric protein known to date that belongs to the [LDH-like] MalDH family. All the other known members of this family are homo-tetramers. The crystal structures revealed that the association of the dimers to form tetramers is prevented by several deletions, taking place at the level of two loops that are known to be essential for the tetramerisation process within the LDH and [LDH-like] MalDH enzymes.

Archaeoglobus fulgidus↗

PubMed2003

Improved 3D continuum calculations of ion flux through membrane channels.

A continuum model, based on the Poisson-Nernst-Planck (PNP) theory, is applied to simulate steady-state ion flux through protein channels. The PNP equations are modified to explicitly account (1) for the desolvation of mobile ions in the membrane pore and (2) for effects related to ion sizes. The proposed algorithm for a three-dimensional self-consistent solution of PNP equations, in which final results are refined by a focusing technique, is shown to be suitable for arbitrary channel geometry and arbitrary protein charge distribution. The role of the pore shape and protein charge distribution in formation of basic electrodiffusion properties, such as channel conductivity and selectivity, as well as concentration distributions of mobile ions in the pore region, are illustrated by simulations on model channels. The influence of the ionic strength in the bulk solution and of the externally applied electric field on channel properties are also discussed.

Algorithms↗

PubMed2003

The catalytic mechanism of Drosophila alcohol dehydrogenase: evidence for a proton relay modulated by the coupled ionization of the active site Lysine/Tyrosine pair and a NAD+ ribose OH switch.

The ionization properties of the active site residues in Drosophila lebanonensis alcohol dehydrogenase (DADH) were investigated theoretically by using an approach developed to account for multiple locations of the hydrogen atoms of the titratable and polar groups. The electrostatic calculations show that (a) the protonation/deprotonation transition of the binary complex of DADH is related to the coupled ionization of Tyr151 and Lys155 in the active site and (b) the pH dependence of the proton abstraction is correlated with a reorganization of the hydrogen bond network in the active site. On this basis, a proton relay mechanism for substrate dehydrogenation is proposed in which the O2' ribose hydroxyl group from the coenzyme has a key role and acts as a switch. The proton relay chain includes the active site catalytic residues, as well as a chain of eight water molecules that connects the active site with the bulk solvent.

Alcohol Dehydrogenase↗

PubMed2003

Human interferon gamma: significance of the C-terminal flexible domain for its biological activity.

The significance of the C-terminal part of human interferon gamma (hIFNgamma) for its biological activity was studied by 3(')-end gene mutagenesis. A series of nine derivative genes obtained by systemic deletion of three codons was constructed and expressed in Escherichia coli LE392. It was shown that the yield of recombinant protein gradually decreased and the solubility gradually increased with truncation of the C terminus. To avoid artifacts related to the imperfect folding of the proteins during purification, the biological activity of the hIFNgamma proteins was measured in clear cell lysates containing the soluble fractions only. The deletion of the C terminus had a two-step effect on both hIFNgamma antiviral and antiproliferative activities. Whereas the removal of the last 3, 6, and 9 C-terminal amino acids led to a gradual increase (up to 10 times) in biological activity of hIFNgamma, the deletion of more than 9 amino acids had an opposite effect. The truncation of the whole unstructured C-terminal domain resulted in a 10-fold decrease (but not in a complete loss) in biological activity of hIFNgamma. The latter was sequestered upon deletion of 24 amino acids, 3 of which belonged to the alpha-helical domain F.

Amino Acid Sequence↗

PubMed2003

Energetics of sequence-specific protein-DNA association: binding of integrase Tn916 to its target DNA.

The DNA binding domain of the transposon Tn916 integrase (INT-DBD) binds to its DNA target site by positioning the face of a three-stranded antiparallel beta-sheet within the major groove. Binding of INT-DBD to a 13 base pair duplex DNA target site was studied by isothermal titration calorimetry, differential scanning calorimetry, thermal melting followed by circular dichroism spectroscopy, and fluorescence spectroscopy. The observed heat capacity change accompanying the association reaction (DeltaC(p)) is temperature-dependent, decreasing from -1.4 kJ K(-1) mol(-1) at 4 degrees C to -2.9 kJ K(-1) mol(-1) at 30 degrees C. The reason is that the partial molar heat capacities of the free protein, the free DNA duplex, and the protein-DNA complex are not changing in parallel when the temperature increases and that thermal motions of the protein and the DNA are restricted in the complex. After correction for this effect, DeltaC(p) is -1.8 kJ K(-1) mol(-1) and temperature-independent. However, this value is still higher than DeltaC(p) of -1.2 kJ K(-1) mol(-1) estimated by semiempirical methods from dehydration of surface area buried at the complex interface. We propose that the discrepancy between the measured and the structure-based prediction of binding energetics is caused by incomplete dehydration of polar groups in the complex. In support, we identify cavities at the interface that are large enough to accommodate approximately 10 water molecules. Our results highlight the difficulties of structure-based prediction of DeltaC(p) (and other thermodynamic parameters) and emphasize how important it is to consider changes of thermal motions and soft vibrational modi in protein-DNA association reactions. This requires not only a detailed investigation of the energetics of the complex but also of the folding thermodynamics of the protein and the DNA alone, which are described in the accompanying paper [Milev et al. (2003) Biochemistry 42, 3492-3502].

Base Sequence↗

PubMed2003

Energetics of sequence-specific protein-DNA association: conformational stability of the DNA binding domain of integrase Tn916 and its cognate DNA duplex.

Sequence-specific DNA recognition by bacterial integrase Tn916 involves structural rearrangements of both the protein and the DNA duplex. Energetic contributions from changes of conformation, thermal motions and soft vibrational modi of the protein, the DNA, and the complex significantly influence the energetic profile of protein-DNA association. Understanding the energetics of such a complicated system requires not only a detailed calorimetric investigation of the association reaction but also of the components in isolation. Here we report on the conformational stability of the integrase Tn916 DNA binding domain and its cognate 13 base pair target DNA duplex. Using a combination of temperature and denaturant induced unfolding experiments, we find that the 74-residue DNA binding domain is compact and unfolds cooperatively with only small deviation from two-state behavior. Scanning calorimetry reveals an increase of the heat capacity of the native protein attributable to increased thermal fluctuations. From the combined calorimetric and spectroscopic experiments, the parameters of protein unfolding are T(m) = 43.8 +/- 0.3 degrees C, DeltaH(m) = 255 +/- 18 kJ mol(-1), DeltaS(m) = 0.80 +/- 0.06 kJ mol(-1), and DeltaC(p) = 5.0 +/- 0.8 kJ K(-1) mol(-1). The DNA target duplex displays a thermodynamic signature typical of short oligonucleotide duplexes: significant heat absorption due to end fraying and twisting precedes cooperative unfolding and dissociation. The parameters for DNA unfolding and dissociation are DeltaH(m) = 335 +/- 4 kJ mol(-1) and DeltaC(p) = 2.7 +/- 0.9 kJ K(-(1) mol(-1). The results reported here have been instrumental in interpreting the thermodynamic features of the association reaction of the integrase with its 13 base pair target DNA duplex reported in the accompanying paper [Milev et al. (2003) Biochemistry 42, 3481-3491].

Base Sequence↗

PubMed2002

Continuum electrostatic analysis of irregular ionization and proton allocation in proteins.

Irregular (nonsigmoidal) ionization behavior of titratable groups in proteins is analyzed theoretically, using a computational algorithm designed to count explicitly for tautomers of titratable groups and different locations of polar hydrogens. On the basis of calculations for different model systems (acid-acid, base-base, acid-base pairs, and cluster of three strongly interacting groups), it is demonstrated that the pK values, extracted from nonsigmoidal titration curves by fitting to a sum of Henderson-Hasselbalch equations, do not describe the ionization equilibrium correctly. The conditions for observation of irregular titration curves are derived analytically for the case of arbitrary couple of interacting ionizable groups. A possible relation between irregularly shaped titration curves and tautomerization is also illustrated. The protonation-deprotonation equilibrium of Asp76 in ribonuclease T1 is shown to be coupled to dipole reorientation of a water molecule bound at the protein-solvent interface. This finding provides a new interpretation of the experimentally observed chemical shift of this residue.

Amino Acids↗

PubMed2002

Modeling of denatured state for calculation of the electrostatic contribution to protein stability.

Existing models of the denatured state of proteins consider only one possible spatial distribution of protein charges and therefore are applicable to a limited number of cases. In this article, a more general framework for the modeling of the denatured state is proposed. It is based on the assumption that the titratable groups of an unfolded protein can adopt a quasi-random distribution restricted by the protein sequence. The model was applied for the calculations of electrostatic interactions in two proteins, barnase and N-terminal domain of the ribosomal protein L9. The calculated free energy of denaturation, DeltaG(pH), reproduces the experimental data better than the commonly used null approximation (NA). It was shown that the seemingly good agreement with experimental data obtained by NA originates from the compensatory effect between the pairwise electrostatic interactions and the desolvation energy of the individual sites. It was also found that the ionization properties of denatured proteins are influenced by the protein sequence.

Bacterial Proteins↗

PubMed2002

Electrostatic properties of the anion selective porin Omp32 from Delftia acidovorans and of the arginine cluster of bacterial porins.

The functional properties of the anion-selective porin Omp32 from the bacterium Delftia acidovorans, formerly Comamonas acidovorans, are determined by the particularly narrow channel constriction and the electrostatic field inside and outside the pore. A cluster of arginines (Arg 38, Arg 75, and Arg 133) determines the electrostatic field close to the constriction zone. Stacked amino acids carrying charges are prone to drastic pK(a) shifts. However, optimized calculations of the titration behavior of charged groups, based on the finite-difference Poisson-Boltzmann technique, suggest that all the arginines are charged at physiological pH. Protonation of the clustered arginines is stabilized by one buried glutamate residue (Glu 58), which is strongly interacting with Arg 75 and Arg 38. This functional arrangement of three charged amino acid residues is of general significance because it is found in the constriction zones of all known 16-stranded porins from the alpha-, beta-, and gamma-proteobacteria.

Amino Acid Sequence↗

PubMedDate not supplied

Fluorescence studies on denaturation and stability of recombinant human interferon-gamma.

Unfolding/folding transitions of recombinant human interferon-gamma (hIFNgamma) in urea and guanidine chloride (Gn.HCl) solutions were studied by fluorescence spectroscopy. At pH 7.4 Gn.HCl was a much more efficient denaturant (midpoint of unfolding C* = 1.1 M and deltaG0 = 13.4 kJ/mol) than urea (C* = 2.8 M and deltaG0 = 11.7 kJ/mol). The close deltaG0 values indicate that the contribution of electrostatic interactions to the stability of hIFNgamma is insignificant. Both the pH dependence of the fluorescence intensity and the unfolding experiments in urea at variable pH showed that hIFNgamma remains native in the pH range of 4.8-9.5. Using two quenchers, iodide and acrylamide, and applying the Stern-Volmer equation, a cluster of acidic groups situated in close proximity to the single tryptophan residue was identified. Based on the denaturation experiments at different pH values and on our earlier calculations of the electrostatic interactions in hIFNgamma, we assume that the protonation of Asp63 causes conformational changes having a substantial impact on the stability of hIFNgamma.

Buffers↗