Folding recombinant spider-silk in H2 O: 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% H2 O 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 H2 O, an important mile-stone for widespread use of recombinant silk polymers.

High-resolution NMR characterization of a spider-silk mimetic composed of 15 tandem repeats and a CRGD motif.

Multidimensional solution NMR spectroscopic techniques have been used to obtain atomic level information about a recombinant spider silk construct in hexafluoro-isopropanol (HFIP). The synthetic 49 kDa silk-like protein mimics authentic silk from Nephila clavipes, with the inclusion of an extracellular matrix recognition motif. 2D (1)H-(15)N HSQC NMR spectroscopy reveals 33 cross peaks, which were assigned to amino acid residues in the semicrystalline repeat units. Signals from the amorphous segments in the primary sequence were weak and broad, suggesting that this region is highly dynamic and undergoing conformational exchange. An analysis of the deviations of the (13)C(alpha), (13)C(beta), and (13)CO chemical shifts relative to the expected random coil values reveals two highly alpha-helical regions from amino acid 12-19 and 26-32, which comprise the polyalanine track and a GGLGSQ sequence. This finding is further supported by phi-value analysis and sequential and medium-range NOE interactions. Pulsed field gradient NMR measurements indicate that the topology of the silk mimetic in HFIP is nonglobular. Moreover, the 3D (15)N-NOESY HSQC spectrum exhibits few long-range NOEs. Similar spectral features have been observed for repeat modules in other polypeptides and are characteristic of an elongated conformation. The results provide a residue-specific description of a silk sequence in nonaqueous solution and may be insightful for understanding the fold and topology of highly concentrated, stable silk before spinning. Additionally, the insights obtained may find application in future design and large-scale production and storage of synthetic silks in organic solvents.

Acid-induced equilibrium folding intermediate of human platelet profilin.

The acid-induced unfolding of human platelet profilin (HPP) can be minimally modeled as a three-state process. Equilibrium unfolding studies have been performed on human platelet profilin1 (HPP) and monitored by far-UV circular dichroism, tryptophan fluorescence, ANS binding, and NMR spectroscopy. Far-UV CD measurements obtained by acid titration demonstrate that HPP unfolds via a three-state mechanism (N --> I --> U), with a highly populated intermediate between pH 4 and 5. Approximately 80% of native helical secondary structural content remains at pH 4, as indicated by monitoring the CD signal at 222 nm. The stability (DeltaGH2O) of the native conformation at pH 7.0 (obtained by monitoring the change in tryptophan signal as a function of urea concentration) is 5.56 +/- 0.51 kcal mol-1; however, the DeltaGH2O for the intermediate species at pH 4 is 2.01 +/- 0.47 kcal mol-1. The calculated m-values for the pH 7.0 and pH 4.0 species were 1.64 +/- 0.15 and 1.34 +/- 0.17 kcal mol-1 M-1, respectively, which is an indication that the native and intermediate species are similarly compact. Additionally, translational diffusion measurements obtained by NMR spectroscopy and ANS binding studies are consistent with a globular and compact conformation at both pH 7.0 and 4.0. The pKa values for the two histidine (His) residues located on helix 4 of HPP were determined to be 5.6 and 5.7 pH units. These pKa values coincide with the midpoint of the far-UV CD acid titration curve and suggest that the protonation of one or both His residues may play a role in the formation of the unfolding intermediate. Stable intermediate species populate the 2D 1H-15N HSQC NMR spectra between pH 4 and 5. A number of backbone and side-chain resonances show significant perturbations relative to the native spectrum; however, considerable nativelike tertiary contacts remain. Interestingly, the residues on HPP that are significantly altered at low pH coincide with segments of the G-actin binding surface and poly-l-proline binding interface. The earlier reports that a decrease in pH below 6.0 induces structural alterations in profilin, favoring dissociation of the profilin-actin complex, corresponds with the structural alterations observed in the partially unfolded species. Our findings suggest that a novel mechanism for pH induced disruption of the profilin-G-actin complex involve a nativelike unfolding intermediate of profilin.