RESUMO
Based upon precise terahertz (THz) measurements of the solvated amino acid glycine and accompanying ab-initio molecular-dynamics simulations, we show that the N-C-C-O open/close mode at 315â cm-1 serves as a sensitive, label-free probe for the local protonation of the amide group. Experimentally, we can show that this holds not only for glycine but also for diglycine and valine. The approach is more general, since the changes due to protonation result in intensity changes which can be probed by THz time domain (0-50â cm-1 ) as well as by precise THz-FT spectroscopy (50-400â cm-1 ). A detailed analysis allows us to directly correlate the titration spectra with pKa values. This demonstrates the potential of THz spectroscopy to probe the charge state of a natural amino acid in water in a label-free manner.
RESUMO
When comparing protein folding in vitro and in vivo significant differences have been found. This has been attributed to crowding and confinement effects. Using a combination of GHz- and THz-dielectric relaxation spectroscopy and MD simulations, we studied hydration dynamics and reviewed protein stability data inside sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and cetyltrimethylammonium bromide (CTAB) reverse micelles which are model systems for confinement. We find that water inside anionic AOT and cationic CTAB reverse micelles is characterized by a strong dielectric depolarization giving rise to a very low relative permittivity compared to an unconfined solution. Despite differences in the hydration dynamics of the surfactant's head groups, simulations using the two-phase thermodynamics method predict a similar reduction in water entropy for both reverse micelle systems compared to bulk water. When we compare the stability data of proteins in these reverse micelles we find that in contrast to our initial expectation, protein stability correlates rather with the local chemistry of the hydrated head groups than with the excluded volume effect or the low global permittivity.
Assuntos
Micelas , Estabilidade Proteica , Tensoativos/química , Espectroscopia Dielétrica , Simulação de Dinâmica Molecular , Água/químicaRESUMO
Remarkably little is known about the mechanism of action of ice nucleation proteins (INPs), although their ability to trigger ice nucleation could be used in a broad variety of applications. We present CD measurements of an insect lipoprotein ice nucleator (LPIN) which show that the lipoproteins consist of a high amount of ß-structures (35%). Terahertz absorption spectroscopy is used to probe the influence of the LPIN on the H-bond network dynamics. We observe a small, but significant THz excess, as an indication of an influence on the H-bond network dynamics. When adding the ice nucleation inhibitor sodium borate, this effect is considerably reduced, similar to that observed before for antifreeze glycoproteins (AFGPs). We propose that myo-inositol, the functional group of phosphatidylinositols, is crucial for the observed change of the H-bond network dynamics of hydration water. This hypothesis is confirmed by additional THz experiments which revealed that the influence of myo-inositol on the hydrogen bond network can be blocked by sodium borate, similar to the case of LPINs. Interestingly, we find a less significant effect when myo-inositol is replaced for chiro- and allo-inositol which underlines the importance of the exact positioning of the OH groups for the interaction with the H-bond network. We propose that a local ordering of water molecules is supporting ice nucleation activity for the LPIN in a similar way to that found for AFP activity in the case of hyperactive insect AFPs.
Assuntos
Proteínas Anticongelantes/química , Inositol/química , Insetos/química , Lipoproteínas/química , Animais , Proteínas da Membrana Bacteriana Externa , Hidrogênio , Ligação de Hidrogênio , Lipoproteínas/metabolismo , ÁguaRESUMO
We use surface-specific vibrational sum-frequency generation spectroscopy (VSFG) to study the structure and self-assembling mechanism of the class I hydrophobin SC3 from Schizophyllum commune and the class II hydrophobin HFBI from Trichoderma reesei. We find that both hydrophobins readily accumulate at the water-air interface and form rigid, highly ordered protein films that give rise to prominent VSFG signals. We identify several resonances that are associated with ß-sheet structures and assign them to the central ß-barrel core present in both proteins. Differences between the hydrophobin classes are observed in their interfacial self-assembly. For HFBI, we observe no changes in conformation upon adsorption to the water surface. For SC3, we observe an increase in ß-sheet-specific signals that supports a surface-driven self-assembly mechanism in which the central ß-barrel remains intact and stacks into a larger-scale architecture, amyloid-like rodlets.