Time‐resolved FTIR difference spectroscopy as tool for investigating refolding reactions of ribonuclease T1 synchronized with trans → cis prolyl isomerization

Abstract

The structurally well-characterized enzyme ribonuclease T1 was used as a model protein to further evaluate time-resolved Fourier transform IR difference spectroscopy in conjunction with temperature-jump techniques as a useful detection technique for protein folding studies. Compared to the wild-type protein, it was confirmed that the lack of one cis-proline bond at position 55 of the S54G/P55N variant is sufficient to significantly simplify and accelerate the refolding process. This result was sustained by the characterization of the early refolding events that occurred within the experimental dead time.

FTIR and nDSC as Analytical Tools for High-Concentration Protein Formulations

“…The T m (FTIR) of the IgG 1 antibody increases in the following order according to the method of data interpretation applied: midpoint of the second-derivative intensity of the temperature-induced structure (method i) e midpoint of the ratio of the intensities of the arising to the vanishing band using the nonmodified amide I spectrum (method iii) < point of cross section between the intensities of the native and denatured frequencies in the second-derivative spectrum (method iv) < midpoint of the second-derivative intensity of the structural loss band (method ii). The differences in the transition temperatures with reference to a distinct secondary structural element are in contradiction to the results of Moritz et al (28), who stated a coincidence in the temperature profiles of a wild-type RNase T1 and its variant independent from the selection of the amide I band. The T m (FTIR) values were derived from different spectral components comprising, on the one hand, arising structures and, on the other hand, vanishing structures.…”

Formation of Critical Oligomers Is a Key Event during Conformational Transition of Recombinant Syrian Hamster Prion Protein

“…However, when PrP was unfolded completely by 6 M GdnHCl at pH 7, the band shifted to 1516 cm Ϫ1 (data not shown). A shift in this range is significant and typical for unfolding and refolding processes of proteins (46,47). This indicates that the alterations of the secondary structure during the transition process did not change the microenvironments of the tyrosine residues as strongly as a complete unfolding procedure did.…”

Cis−Trans Isomerization of Organic Molecules and Biomolecules: Implications and Applications

“…450 Mutagenesis of proline residues also provide important information about folding kinetics that might implicate particular proline residues; however, the results must be confirmed by complementary techniques. 451 In most cases, cis-trans isomerism is significantly faster than methodologies employed for the purification of molecules (i.e., HPLC) and does not enable the separation of isomers. However, there are some examples of peak splitting (i.e., erythropoietin, 452 enalapril) 453 and peak separation either at low 434 or room temperature.…”