Salt effects on hydrophobic‐core formation in folding of a helical miniprotein studied by molecular dynamics simulations

We have investigated effects of salt ions on folding events of a helical miniprotein chicken villin headpiece subdomain HP36. Low concentrations of ions alter electrostatic interactions between charged groups of a protein and can change the populations of conformers. Here, we compare two data sets of folding simulations of HP36 in explicit water solvent with or without ions. For efficient sampling of the conformational space of HP36, the multicanonical replica‐exchange molecular dynamics method was employed. Our analyses suggest that salt alters salt‐bridging nature of the protein at later stages of folding at room temperature. Especially, more nonnative, nonlocal salt bridges are formed at near‐native conformations in pure water. Our analyses also show that such salt‐bridge formation hinders the fully native hydrophobic‐core packing at the final stages of folding. Proteins 2014; 82:933–943. © 2013 Wiley Periodicals, Inc.     

Tat cell-penetrating peptide has the characteristics of a poly(proline) II helix in aqueous solution and in SDS micelles.

Tat cell-penetrating peptide (GRKKRRQRRRPPQG) is able to translocate and carry molecules across cell membranes. Using CD spectroscopy the conformation of this synthetic peptide was studied in aqueous and membrane-mimicking, micellar SDS solutions at different temperatures. The CD spectrum of the Tat cell-penetrating peptide in SDS micellar solution was virtually unchanged from that in aqueous solution, and at low temperature it was close to that of a poly(proline) II helix.     

Folding recombinant spider‐silk in H2O: Effect of osmolytes on the solution conformation of a 15‐repeat spider‐silk mimetic

The folding of a recombinant spider silk protein‐polymer in the presence of the tri‐methylamine osmolytes TMANO and Betaine in 80% H₂O is reported. Circular dichroism measurements (CD) reveal an increase in α‐helical secondary structure with increasing osmolyte concentrations, as determined by an increase in ellipticity at 222 nm. Consistent with this observation, the signal for random coil sampling, observed at 205 nm, is greatly reduced with increasing trimethylamine. Fluorescence spectra of a single tyrosine positioned within the conserved 33‐amino acid repeat primary sequence (of the spider‐silk mimetic) complements the conformational changes observed by CD. Importantly, there is a correlation between the number of Alkyl‐groups (CH₃‐) on the amine of the osmolyte and enhanced helicity of the 15‐repeat silk‐mimetic for the osmolytes tested, ie TMANO, Betaine, Sarcosine and Glycine. These preliminary results are applicable to storing and processing recombinant silk sequences in H₂O, an important mile‐stone for widespread use of recombinant silk polymers.     

Vibrational circular dichroism of a 2,5‐diketopiperazine (DKP) peptide: Evidence for dimer formation in cyclo LL or LD diphenylalanine in the solid state

The diastereomer diketopiperazine (DKP) peptides built on phenylalanine, namely, cyclo diphenylalanine LPhe‐LPhe and LPhe‐DPhe, were studied in the solid phase by vibrational circular dichroism (VCD) coupled to quantum chemical calculations. The unit structure of cyclo LPhe‐LPhe in KBr pellets is a dimer bridged by two strong NH…O hydrogen bonds. The intense bisignate signature in the CO stretch region is interpreted in terms of two contributions arising from the free COs of the dimer and the antisymmetrical combination of the bound COs. In contrast, cyclo LPhe‐DPhe shows no VCD signal in relation to its symmetric nature.     

Reversibly bound chloride in the atrial natriuretic peptide receptor hormone‐binding domain: Possible allosteric regulation and a conserved structural motif for the chloride‐binding site

The binding of atrial natriuretic peptide (ANP) to its receptor requires chloride, and it is chloride concentration dependent. The extracellular domain (ECD) of the ANP receptor (ANPR) contains a chloride near the ANP‐binding site, suggesting a possible regulatory role. The bound chloride, however, is completely buried in the polypeptide fold, and its functional role has remained unclear. Here, we have confirmed that chloride is necessary for ANP binding to the recombinant ECD or the full‐length ANPR expressed in CHO cells. ECD without chloride (ECD(?)) did not bind ANP. Its binding activity was fully restored by bromide or chloride addition. A new X‐ray structure of the bromide‐bound ECD is essentially identical to that of the chloride‐bound ECD. Furthermore, bromide atoms are localized at the same positions as chloride atoms both in the apo and in the ANP‐bound structures, indicating exchangeable and reversible halide binding. Far‐UV CD and thermal unfolding data show that ECD(?) largely retains the native structure. Sedimentation equilibrium in the absence of chloride shows that ECD(?) forms a strongly associated dimer, possibly preventing the structural rearrangement of the two monomers that is necessary for ANP binding. The primary and tertiary structures of the chloride‐binding site in ANPR are highly conserved among receptor‐guanylate cyclases and metabotropic glutamate receptors. The chloride‐dependent ANP binding, reversible chloride binding, and the highly conserved chloride‐binding site motif suggest a regulatory role for the receptor bound chloride. Chloride‐dependent regulation of ANPR may operate in the kidney, modulating ANP‐induced natriuresis.     

Insights into the binding specificity of wild type and mutated wheat germ agglutinin towards Neu5Acα(2‐3)Gal: a study by in silico mutations and molecular dynamics simulations

Wheat germ agglutinin (WGA) is a plant lectin, which specifically recognizes the sugars NeuNAc and GlcNAc. Mutated WGA with enhanced binding specificity can be used as biomarkers for cancer. In silico mutations are performed at the active site of WGA to enhance the binding specificity towards sialylglycans, and molecular dynamics simulations of 20 ns are carried out for wild type and mutated WGAs (WGA1, WGA2, and WGA3) in complex with sialylgalactose to examine the change in binding specificity. MD simulations reveal the change in binding specificity of wild type and mutated WGAs towards sialylgalactose and bound conformational flexibility of sialylgalactose. The mutated polar amino acid residues Asn114 (S114N), Lys118 (G118K), and Arg118 (G118R) make direct and water mediated hydrogen bonds and hydrophobic interactions with sialylgalactose. An analysis of possible hydrogen bonds, hydrophobic interactions, total pair wise interaction energy between active site residues and sialylgalactose and MM‐PBSA free energy calculation reveals the plausible binding modes and the role of water in stabilizing different binding modes. An interesting observation is that the binding specificity of mutated WGAs (cyborg lectin) towards sialylgalactose is found to be higher in double point mutation (WGA3). One of the substituted residues Arg118 plays a crucial role in sugar binding. Based on the interactions and energy calculations, it is concluded that the order of binding specificity of WGAs towards sialylgalactose is WGA3 > WGA1 > WGA2 > WGA. On comparing with the wild type, double point mutated WGA (WGA3) exhibits increased specificity towards sialylgalactose, and thus, it can be effectively used in targeted drug delivery and as biological cell marker in cancer therapeutics. Copyright © 2014 John Wiley & Sons, Ltd.