Pressure-Induced Unfolding/Refolding of Ribonuclease A:  Static and Kinetic Fourier Transform Infrared Spectroscopy Study

Abstract

In this paper, we illustrate the use of high-pressure Fourier transform infrared (FT-IR) spectroscopy to study the reversible presssure-induced unfolding and refolding of ribonuclease A (RNase A) and compare it with the results obtained for the temperature-induced transition. FT-IR spectroscopy monitors changes in the secondary structural properties (amide I' band) or tertiary contacts (tyrosine band) of the protein upon pressurization or depressurization. Analysis of the amide I' spectral components reveals that the pressure-induced denaturation process sets in at 5. 5 kbar at 20 degrees C and pH 2.5. It is accompanied by an increase in disordered structures while the content of beta-sheets and alpha-helices drastically decreases. The denatured state above 7 kbar retains nonetheless some degree of beta-like secondary structure and the molecule cannot be described as an extended random coil. Increase of pH from 2.5 to 5.5 has no influence on the structure of the pressure-denatured state; it slightly changes the stability of the protein only. All experimental evidence indicates that the pressure-denatured states of monomeric proteins have more secondary structure than the temperature-denatured states. Different modes of denaturation, including pressure, may correlate differently with the roughness of the energy scale and slope of the folding funnel. For these reasons we have also carried out pressure-jump kinetic studies of the secondary structural evolution in the unfolding/refolding reaction of RNase A. In agreement with the theoretical model presented by Hummer et al. [(1998) Proc. Natl. Acad. Sci. U.S.A. 95, 1552-1555], the experimental data show that pressure slows down folding and unfolding kinetics (here 1-2 orders of magnitude), corresponding to an increasingly rough landscape. The kinetics remains non-two-state under pressure. Assuming a two-step folding scenario, the calculated relaxation times for unfolding of RNase A at 20 degrees C and pH 2.5 can be estimated to be tau(1) approximately 0.7 min and tau(2) approximately 17 min. The refolding process is considerably faster (tau(1) approximately 0.3 min, tau(2) approximately 4 min). Our data show that the pressure stability and pressure-induced unfolding/refolding kinetics of monomeric proteins, such as wild-type staphylococcal nuclease (WT SNase) and RNase A, may be significantly different. The differences are largely due to the four disulfide bonds in RNase A, which stabilize adjacent structures. They probably lead to the much higher denaturation pressure compared to SNase, and this might also explain why the volume change of WT SNase upon unfolding is about twice as large.

Molecular View by Fourier Transform Infrared Spectroscopy of the Relationship between Lactocin 705 and Membranes: Speculations on Antimicrobial Mechanism

“…This fact is proved by the decrease of the conformational order of the DPPC acyl chains without significant change in the membrane interfacial region upon Lac705␤ peptide addition (41). The peptide's tyrosine wave number shifting from 1,515.14 cm Ϫ1 to 1,511.5 cm Ϫ1 is also evidence of the peptide's change to a more hydrophobic environment (9,38). These results support the hypothesis that this peptide could insert into the DPPC bilayer (22).…”

“…However, this wave number is affected by the tyrosine's local environment being different according to whether it is exposed to water or to a hydrophobic environment. For example, it increases upon peptide unfolding (22,38). Therefore, this vibration mode may represent a marker providing information relative to the peptide surroundings (9).…”

Thermodynamic theory of nonlinear ultrasound in soft biological tissue

“…According to equations (2.15), we can write 18) where, in agreement with the first equation of system (2.11), the function R t on the right-hand side of equation (2.15) has been expressed in terms of U x . The total relaxation pressure I p corresponding to the infinite number of degrees of freedom characterized by the distribution…”

“…The existing evidence [18,19] suggests that the denatured protein is not a single uniform state, but is a large statistical ensemble of molecular conformation states with similar free energies. These conformation states can rapidly establish equilibrium with one another, and are highly sensitive to thermodynamic perturbations of the medium.…”

Pressure Induced Structural Changes and Gas Diffusion Pathways in Monomeric Fluorescent Proteins

“…Pressure experiments on proteins have shown that proteins can be squeezed (6)(7)(8)(9)(10)(11) (14,15). Investigations of the effects on the protein structure of gradually increasing the pressure help in understanding the mechanistic details about protein functioning and interactions between proteins and solvents (7,16,17).…”