Vladimir V. Filimonov

A thermodynamic analysis of a family of small globular proteins: SH3 domains

The stability and folding thermodynamics of two SH3-domains, belonging to Fyn and Abl proteins, have been studied by scanning calorimetry and urea-induced unfolding. They undergo an essentially two-state unfolding with parameters similar to those of the previously studied alpha-spectrin SH3 domain. The correlations between the thermodynamic parameters (heat capacity increment, delta Cp,U, the proportionality factor, m, and the Gibbs energy, delta Gw298) of unfolding and some integral structural parameters, such as polar and non-polar areas exposed upon domain denaturation, have been analyzed. The experimental data on delta Cp,U and the m-factor of the linear extrapolation model (LEM) obey the simple empirical correlations deduced elsewhere. The Gibbs energies calculated from the DSC data were compared with those found by fitting urea-unfolding curves to the LEM and the denaturant-binding model (DBM). The delta Gw298 values found with DBM correlate better with the DSC data, while those obtained with LEM are systematically smaller. The systematic difference between the parameters calculated with LEM and DBM are explained by an inherent imperfection of the LEM.

A Thermodynamic and Kinetic Analysis of the Folding Pathway of an SH3 Domain Entropically Stabilised by a Redesigned Hydrophobic Core

The folding thermodynamics and kinetics of the alpha-spectrin SH3 domain with a redesigned hydrophobic core have been studied. The introduction of five replacements, A11V, V23L, M25V, V44I and V58L, resulted in an increase of 16% in the overall volume of the side-chains forming the hydrophobic core but caused no remarkable changes to the positions of the backbone atoms. Judging by the scanning calorimetry data, the increased stability of the folded structure of the new SH3-variant is caused by entropic factors, since the changes in heat capacity and enthalpy upon the unfolding of the wild-type and mutant proteins were identical at 298 K. It appears that the design process resulted in an increase in burying both the hydrophobic and hydrophilic surfaces, which resulted in a compensatory effect upon the changes in heat capacity and enthalpy. Kinetic analysis shows that both the folding and unfolding rate constants are higher for the new variant, suggesting that its transition state becomes more stable compared to the folded and unfolded states. The phi(double dagger-U) values found for a number of side-chains are slightly lower than those of the wild-type protein, indicating that although the transition state ensemble (TSE) did not change overall, it has moved towards a more denatured conformation, in accordance with Hammonds postulate. Thus, the acceleration of the folding-unfolding reactions is caused mainly by an improvement in the specific and/or non-specific hydrophobic interactions within the TSE rather than by changes in the contact order. Experimental evidence showing that the TSE changes globally according to its hydrophobic content suggests that hydrophobicity may modulate the kinetic behaviour and also the folding pathway of a protein.

AS-48: a circular protein with an extremely stable globular structure

The unfolding thermodynamics of the circular enterocin protein AS‐48, produced by Enterococcus faecalis, has been characterized by differential scanning calorimetry. The native structure of the 70‐residue protein is extremely thermally stable. Thus, at pH 2.5 and low ionic strength thermal denaturation occurs under equilibrium at 102°C, while the unfolded state irreversibly aggregates at neutral and alkaline pH. Calorimetric data analysis shows that the specific enthalpy change upon unfolding is unusually small and the heat capacity change is quite normal for a protein of this size, whereas the Gibbs energy change at 25°C is relatively high. At least part of this high stability might be put down to entropic constraints induced by the circular organization of the polypeptide chain.

Flagellin parts acquiring a regular structure during polymerization are disposed on the molecule ends

Flagellins of two Escherichia coli strains have been investigated by limited proteolysis and scanning microcalorimetry. It has been shown that a monomer flagellin consists of two parts: a central one cooperatively melting, rather resistant to proteases, and the other without a stable tertiary structure and thus easily degrading terminals. Just these terminals acquire a regular structure during polymerization. Core fragments of the two strains have been isolated and characterized.

STRUCTURAL ANALYSIS OF THE A AND B CONFORMERS OF ESCHERICHIA COLI 5 S RIBOSOMAL RNA BY INFRARED SPECTROSCOPY

1. Introduction Rapid progress is being made in elucidating the structure of pro- and eukaryotic 5 S RNAs using dif- ferent physical, biochemical and sequence analysis approaches (reviewed in [ 11). It seems to be evident from comparative sequence studies [l-5] that a general base pairing scheme of the type first proposed in [2] is valid for eukaryotic 5 S RNAs and that a general secondary structure of the type first proposed in [3] extended by few base pairs is the structural basis for prokaryotic 5 S RNAs. Experimental evi- dence supporting these basic secondary structures is now manifold and derives, e.g., from such powerful specific techniques as high-resolution ‘H NMR spec- troscopy [6,7] and slow tritium exchange studies [8]. Summarizing data from optical ([6,9] and references within), infrared [lo], Raman [l l-131, and ‘H NMR spectroscopy [6,7] it became obvious that both for prokaryotic and eukaryotic 5 S RNA molecules in solution a highly ordered secondary/tertiary structure exists with an amount of about 35-42 base pairs (58-70% of all nucleotides are base-paired) in the presence of stabilizing ions. and the infrared thermal melting curves reveal partic- ular differences between both conformers at 570°C which may be useful with respect to the analysis of the intricate problem of the A-to-B conformational transition(s). 2. Materials and methods Here, we report the results of infrared spectro- scopic studies of the A and B conformers of

The denaturation of circular enterocin AS-48 by urea and guanidinium hydrochloride.

The unfolding thermodynamics of the circular enterocin protein AS-48, produced by Enterococcus faecalis, has been studied. The native structure of the 70-amino-acid-long protein turned out to be extremely stable against heat and denaturant-induced unfolding. At pH 2.5 and low ionic strength, it denatures at 102 degrees C, while at 25 degrees C, the structure only unfolds in 6.3 M guanidinium hydrochloride (GuHCl) and does not unfold even in 8 M urea. A comparison of its thermal unfolding in water and in the presence of urea shows a good correspondence between the two deltaGw(298) values, which are about 30 kJ mol(-1) at pH 2.5 and low ionic strength. The stability of the structure is highly dependent upon ionic strength and so GuHCl acts both as a denaturant and a stabilising agent. This seems to be why the deltaGw(298) value calculated from the unfolding data in GuHCl is twice as high as in the absence of this salt. At least part of the high stability of native AS-48 can almost certainly be put down to its circular organization since other structural features are quite normal for a protein of this size.

Stability of Natrialba magadii NDP kinase: comparisons with other halophilic proteins.

Abstract. Nucleoside diphosphate kinase from the haloalkaliphilic archaeon Natrialba magadii (Nm NDPK) is a homooligomeric hexamer with a monomer molecular weight of 23xa0kDa. Its main function is to exchange γ-phosphates between nucleoside triphosphates and diphosphates. Previously it was shown that Nm NDPK is active over a wide range of NaCl concentrations, which is not typical of extremely halophilic proteins. In this paper more detailed investigations of kinase function and stability were carried out using circular dichroism, differential scanning calorimetry, size-exclusion chromatography, and biochemical methods. A possible mechanism for stabilization of halophilic proteins that allows them to function in a wide range of NaCl concentrations is proposed.

Solution structure and dynamics of the chimeric SH3 domains, SHH- and SHA- Bergeracs

Two chimeric proteins, SHcapital EN, Cyrillic and SHA of the SH3-Bergerac family (where the beta-turn N47D48 in spectrin SH3 domain was substituted for KITVNGKTYE or KATANGKTYE sequences, respectively), were analyzed by high-resolution NMR to resolve their spatial structures and to analyze their dynamics. Although the presence of a stable beta-hairpin in the region of the insertion was confirmed, the introduced extension of the polypeptide chain in SHcapital EN, Cyrillic (approximately 17%) practically did not affect the total molecule topology. Interestingly, the introduced beta-hairpin had higher mobility in comparison with other protein regions. Finally, we performed a disorder prediction with the PONDR VSL2 algorithm and discovered that the inserted beta-hairpin in both SHH and SHA proteins exhibited significant propensity for intrinsic disorder and therefore for high mobility. In agreement with the experimental data, the predisposition for the increased intramolecular mobility was noticeably higher in SHA.

A binding event converted into a folding event

We have designed a chimeric protein by connecting a circular permutant of the α‐spectrin SH3 domain to the proline‐rich decapeptide APSYSPPPPP with a three‐residue link. Our aim was to obtain a single‐chain protein with a tertiary fold that would mimic the binding between SH3 domains and proline‐rich peptides. A comparison of the circular‐dichroism and fluorescence spectra of the purified chimera and the SH3 circular permutant showed that the proline‐rich sequence occupies the putative SH3 binding site in a similar conformation and with comparable interactions to those found in complexes between SH3 and proline‐rich peptides. Differential scanning calorimetry indicated that the interactions in the binding motif interface are highly cooperative with the rest of the structure and thus the protein unfolds in a two‐state process. The chimera is more stable than the circular permutant SH3 by 6–8 kJ mol−1 at 25°C and the difference in their unfolding enthalpy is approximately 32 kJ mol−1, which coincides with the values found for the binding of proline‐rich peptides to SH3 domains. This type of chimeric protein may be useful in designing SH3 peptide ligands with improved affinity and specificity.

The major mRNP protein YB-1: Structural and association properties in solution

YB-1 is a major mRNP protein participating in the regulation of transcription and translation of a wide range of eukaryotic genes in many organisms probably due to its influence on mRNA packing into mRNPs. While the functional properties of YB-1 are extensively studied, little is known about its structural properties. In the present work we focused on studying its secondary structure, rigidity of its tertiary structure, compactness, and oligomerization in vitro by using far UV-CD, DSC, one-dimensional (1)H NMR, SAXS, sedimentation and FPLC. It was shown that only the cold shock domain within the entire YB-1 chain has a well-packed tertiary structure undergoing cooperative heat and cold denaturation transitions. In contrast, the rest of the YB-1 molecule is not rigidly packed and consists of PP II-like helical secondary structure elements and coil-like regions. At the same time, the overall dimension of the protein molecule is unexpectedly small. The polypeptide chains of YB-1 have a high tendency to form oligomers at neutral pH, while the extent and structural organization of the oligomers depend on protein concentration and ionic strength varying from compact monomeric units up to high molecular weight oligomers. These oligomers in solution are unstable and dissociate upon protein concentration decrease.