Abstract We have compared structures of 78 proteins determined by both NMR and X‐ray methods. It is shown that the same protein more differences than various obtained for protein, even protein. 18 these obvious large‐scale structural seem to reflect a difference crystal solution structures. The other 60 pairs only small‐scale comparable with between or protein; we analyzed attentively. One main concerns number contacts per residue: (1) presented in PDB at distances below 3.0 Å 4.5–6.5 Å,...

We present here a simple approach to identify domain boundaries in proteins of an unknown three-dimensional structure. Our method is based on the hypothesis that high-side chain entropy region protein must be compensated by high-residue interaction energy within region, which could correlate with well-structured part globule, is, unit. For domains, this means boundary conditioned amino acid residues small value side entropy, correlates size. On one hand, relatively high Ala and Gly content...

Abstract The most attractive and methodologically convenient way to enhance protein stability is via the introduction of disulphide bond(s). However, effect artificially introduced SS-bond on often quite unpredictable. This raises question how choose sites in an intelligent manner, so that 'fastening' these by SS-bond(s) would provide maximal stability. We hypothesize successful design a stabilizing requires finding highly mobile regions. Using GFP as illustrative example, we demonstrate...

YB-1, a multifunctional DNA- and RNA-binding nucleocytoplasmic protein, is involved in the majority of mRNA-dependent events cell. It consists three structurally different domains: its central cold shock domain has structure β-barrel, while flanking domains are predicted to be intrinsically disordered. Recently, we showed that YB-1 capable forming elongated fibrils under high ionic strength conditions. Here report it responsible for formation fibrils, terminal differentially modulate this...

When considering protein folding with a transient intermediate, difficulty arises as to determination of the rates separate transitions. Here we overcome this problem, using kinetic studies unfolding/refolding reactions three-state apomyoglobin model. Amplitudes refolding burst phase corresponding transition from unfolded (U) intermediate (I) state, that occurs prior native state (N) formation, allow us estimate relative populations rapidly converting states at various final urea...

Abstract Background The majority of experimentally determined crystal structures Type II restriction endonucleases (REases) exhibit a common PD-(D/E)XK fold. Crystal have been also for single representatives two other folds: PLD (R.BfiI) and half-pipe (R.PabI), bioinformatics analyses supported by mutagenesis suggested that some REases belong to the HNH Our previous bioinformatic analysis REase R.Eco29kI shares sequence similarities with one more unrelated nuclease superfamily, GIY-YIG,...

The bacterial Sm-like protein Hfq forms homohexamers both in solution and crystals. monomers are organized as a continuous beta-sheet passing through the whole hexamer ring with common hydrophobic core. Analysis of Pseudomonas aeruginosa (PaeHfq) structure suggested that solvent-inaccessible intermonomer hydrogen bonds created by conserved amino-acid residues should also stabilize quaternary protein. In this work, one such residue, His57, PaeHfq was replaced alanine, threonine or asparagine....

Influence of 12 nonpolar amino acids residues from the hydrophobic core apomyoglobin on stability its native state and folding intermediate was studied. Six selected are A, G H helices; these conserved in structure globin family, although nonfunctional, that is, not involved heme binding. The rest nonconserved belong to B, C, D, E helices. Each residue substituted by alanine, equilibrium pH-induced transitions mutants were studied circular dichroism fluorescent spectroscopy. obtained results...

Is it possible to compare the physicochemical properties of a wild-type protein and its mutant form under same conditions? Provided mutation has destabilized protein, may be more correct native conditions with small amount denaturant. In general, is appropriate proteins by different treatments: mutations, pH, temperature, denaturants like urea? These issues have compelled us search for methods ways presentation experimental results that would allow comparison forms lead conclusions on effect...

The analysis of the three-dimensional structure green fluorescent protein (GFP-cycle3) revealed presence two well-defined hydrophobic clusters located on opposite sides GFP β-can that might contribute to formation partially folded intermediate(s) during unfolding. microcalorimetric nonequilibrium melting GFP-cycle3 and its mutants, I14A I161A, due sequential mentioned clusters, temperature-induced denaturation this most likely occurs in three stages. first second stages involve a smaller...

The most complex problem in studying multi-state protein folding is the determination of sequence formation intermediate states. A far more issue to determine at what stages its various parts (secondary structure elements) develop. and properties different states depend particular on these parts. An experimental approach, named μ-analysis, which allows understanding order structural elements upon a was used this study. In approach same secondary are "tested" by substitutions single...

Bacillus cereus is the fourth most common cause of foodborne illnesses that produces a variety pore-forming proteins as main pathogenic factors. B. hemolysin II (HlyII), belonging to β-barrel toxins, has C-terminal extension 94 amino acid residues designated HlyIICTD. An analysis panel monoclonal antibodies recombinant HlyIICTD protein revealed ability antibody HlyIIC-20 inhibit HlyII hemolysis. A conformational epitope recognized by was found. method peptide phage display and found it...

Bacillus cereus hemolysin II, a pore-forming β-barrel toxin (HlyII), has C-terminal extension of 94 amino acid residues, designated as the domain HlyII (HlyIICTD). HlyIICTD is capable forming oligomers in aqueous solutions. Oligomerization significantly increased presence erythrocytes and liposomes. Its affinity for various origins differed insignificantly but was noticeably higher T-cells. destroyed THP-1 monocytes J774 macrophages, acted most effectively on Jurkat T-lymphocytes had...

Green fluorescent protein (GFP) has been studied quite thoroughly, however, up to now some experimental data have not explained explicitly. For example, under native conditions this can two isoforms differing in their mobility gel. In case, no differences between the are revealed denaturing conditions. order understand difference of protein, we investigated GFP-cycle3 using mass spectrometry, gel electrophoresis, size exclusion chromatography, microcalorimetry, and spectroscopy methods...

Directed stabilization of globular proteins via substitution a minimal number amino acid residues is one the most complicated experimental tasks. This work summarizes our research on effect substitutions protein stability utilizing outputs analysis intrinsic disorder predisposition target proteins. allowed us to formulate basis possible approaches The idea quite simple. To stabilize as whole, needs find its "weakest spot" and it, but question how this weak spot can be found in query protein....

Hemolysin II (HlyII)—one of the pathogenic factors Bacillus cereus, a pore-forming β-barrel toxin—possesses C-terminal extension 94 amino acid residues, designated as domain HlyII (HlyIICTD), which plays an important role in functioning toxin. Our previous work described monoclonal antibody (HlyIIC-20), capable strain-specific inhibition hemolysis caused by HlyII, and demonstrated dependence efficiency on presence proline at position 324 outside conformational antigenic determinant. In this...

Abstract Enhancing protein stability holds paramount significance in biotechnology, therapeutics, and the food industry. Circular permutations offer a distinctive avenue for manipulating while keeping intra-protein interactions intact. Amidst creation of circular permutants, determining optimal placement new N- C-termini stands as pivotal, albeit largely unexplored, endeavor. In this study, we employed PONDR-FIT’s predictions disorder propensity to guide design permutants GroEL apical domain...