Generalized Two-dimensional Correlation Spectroscopic Studies On The Structural Changes Of Proteins Induced By Environmental Variations

Generalized two-dimensional correlation spectroscopy has been extensively used in the study of the secondary structural changes of protein induced by some typical environmental perturbations. The "sequential order" rules of this analytical technique were employed in these studies. It was found that the secondary structural changes of proteins are not in a fully cooperative way but with sequential order. However, debates on the two major advantages of this technique have lasted for more than a decade, including our continuous efforts, which indicated that the new bands resolved by 2D correlation analysis may be not real and the sequence of the intensity variations deduced from the "sequential order" rules may be contradictory to the experimental observation.In this work, generalized two-dimensional correlation spectroscopy and in situ spectroscopy analysis were used to investigate the structural changes of proteins induced by some typical environmental perturbations and test the two major advantages of 2D correlation spectroscopy. The findings may be helpful to the improvement and modification of the generalized two-dimensional correlation spectroscopy.The main contents of this study are as follows:1 The adsorption process of human serum albumin (HSA) on an ATR (ZnSe) crystal was studied. The intensity of the 1650 cm-1 band changes before 1625 cm-1 and 1681 cm-1, i.e.,1650>1625≈1681 cm-1, which is obtained from in situ ATR-FTIR spectra analysis. The adsorption process of HSA on the ATR (ZnSe) crystal in aqueous solutions can be divided into three phases:the adsorption of HSA molecules without obvious conformational transitions in the first 25 min; large structural rearrangement from 25 to 50 min of adsorption when the loss ofα-helix structure is essentially transformed into random coil and short extended chain structures in a fully cooperative way; and further slight conformational transformation of short extended chain and turn structures into random coil with no sequential order after 50 min of adsorption.2 The time-dependent adsorption behavior ofβ-Lg on ZnSe crystal surface was studied by in situ ATR/FTIR and 2D correlation analysis. The intensities of the three bands at 1627,1651 and 1684 cm-1 changed together but before that of 1666 cm-1 band, which is deduced from in situ ATR-FTIR spectra analysis. In the first 15 min of the adsorption time, the change rate of the intensity of three bands in amide I band is 1627 (7.38E-4)>1651 (6.83E-4)>1684 (3.21E-4) cm-1. It can be found from analysis of band area percentages that there were no secondary structural changes in the first 10 min of adsorption ofβ-Lg molecules. From about 10 min to 15 min, the content of antiparallelβ-strands (low-wavenumber component at 1627 cm-1) decreased and was simultaneously transformed into random segments (1651 cm-1). The content ofβ-turns (1666 cm-1) started to decrease, and the loss ofβ-turns (1666 cm-1) was essentially transformed into antiparallelβ-strands (high-wavenumber component at 1684 cm-1) also in a cooperative way.3 Two quenching agents (NaI, acrylamide (AA)) were used to study the quenching process of two tryptophan (Trp) residues in myoglobin from equine heart. The experimental finding obtained from in situ spectroscopy analysis is that the intensities of W7 (350 nm) and W14 (328 nm) change in a cooperative way, without sequential order. It was also found from in situ fluorescence spectroscopy analysis that, AA can quench both W7 and W14 with a very similar rate, however, the intensity of W7 decreases faster than that of W14 when quenched by Nal. It can be found by in situ FTIR spectra analysis that partial unfolding take place when the concentration of HM solution is very low. In the present experimental condition (10μM), the surrounding of W14 is not so hydrophobic as it in higher concentration, so W14 become susceptible to the quencher I-In our studies, the sequential order of the intensity variations of the subbands obtained from the generalized 2D correlation spectroscopy is contradictory to the in situ spectra analysis.