Terahertz underdamped vibrational motion governs protein-ligand binding in solution

Low-frequency collective vibrational modes in proteins have been proposed as being responsible for efficiently directing biochemical reactions and biological energy transport. However, evidence of the existence of delocalized vibrational modes is scarce and proof of their involvement in biological function absent. Here we apply extremely sensitive femtosecond optical Kerr-effect spectroscopy to study the depolarized Raman spectra of lysozyme and its complex with the inhibitor triacetylchitotriose in solution. Underdamped delocalized vibrational modes in the terahertz frequency domain are identified and shown to blue-shift and strengthen upon inhibitor binding. This demonstrates that the ligand-binding coordinate in proteins is underdamped and not simply solvent-controlled as previously assumed. The presence of such underdamped delocalized modes in proteins may have significant implications for the understanding of the efficiency of ligand binding and protein–molecule interactions, and has wider implications for biochemical reactivity and biological function

Water Dynamics at Protein Interfaces: Ultrafast Optical Kerr Effect Study

The behavior of water molecules surrounding a protein can have an important bearing on its structure and function. Consequently, a great deal of attention has been focused on changes in the relaxation dynamics of water when it is located at the protein surface. Here we use the ultrafast optical Kerr effect to study the H-bond structure and dynamics of aqueous solutions of proteins. Measurements are made for three proteins as a function of concentration. We find that the water dynamics in the first solvation layer of the proteins are slowed by up to a factor of 8 in comparison to those in bulk water. The most marked slowdown was observed for the most hydrophilic protein studied, bovine serum albumin, whereas the most hydrophobic protein, trypsin, had a slightly smaller effect. The terahertz Raman spectra of these protein solutions resemble those of pure water up to 5 wt % of protein, above which a new feature appears at 80 cm–1, which is assigned to a bending of the protein amide chain

Adhesion of alumina surfaces through confined water layers containing various molecules

When two surfaces confine water layers between them at the nanoscale, the behaviour of these confined water molecules can deviate significantly from the behaviour of bulk water and it could reflect on the adhesion of such surfaces. Thus, the aim of this study is to assess the role of confined water layers on the adhesion of hydrophilic surfaces and how sensitive this adhesion is to the presence of contaminants. Our methodology used under water AFM force measurements with an alumina sputtered sphere-tipped cantilever and a flat alumina single crystal, then added fractions of ethanol, dimethylformamide, formamide, trimethylamine, and trehalose to water, as contaminants. Such solutions were designed to illuminate the influences of dielectric constant, molecular size, refractive index and number of hydrogen bonds from donors and acceptors of solutes to water. Apart from very dilute solutions of dimethylformamide, all solutions decreased the ability of confined water to give adhesion of the alumina surfaces. The predicted theoretical contribution of van der Waals and electrostatic forces was not observed when the contaminants distorted the way water organizes itself in confinement. The conclusion was that adhesion was sensitive mostly to hydrogen bonding network within water layers confined by the hydrophilic alumina surfaces

Synthetic glycopolymers as modulators of protein aggregation: influence of chemical composition, topology and concentration

Novel drug excipients are required to achieve stable formulations of protein drug candidates. Synthetic glycopolymers have been shown in some cases to improve protein formulation stability, although their structure function relationship remains unknown. Here we report the synthesis of linear or 4-arm star glycopolymers with different molecular topology and chemical composition, with mannose, galactose, arabinose, N-acetyl glucosamine, lactose and trehalose pendant units - and investigate their modulation of conformational stability and aggregation propensity of a model monoclonal antibody (mAb1). Mono-and di-saccharides with free reducing ends are not frequently utilised as protein stabilisers, due to potential reactivity with a protein’s amine groups. In this study this was circumvented through the use of a stable acetal linker connecting the polymer backbone to the sugar pendant residues, which made the latter virtually non-reactive with amines. The general destabilisation the antibody was determined as anunfolding transition temperature (Tm) of CH2 and Fab structural domains, and aggregation temperature (Tagg). The most prominent effect of the glycopolymers on a temperature induced stress in a low concentration solutions was a decrease in Tm and Tagg, regardless of sugar composition or glycopolymer topology - in contrast to the stabilising effect of the corresponding mono- and di-saccharide constituents. The exceptions of linear-lactose and star-trehalose glycopolymers, which increased Tm of the mAb Fab region and Tagg, however, highlights a more complex structure-function relationship. Accelerated stability studies of the high concentrated mAb solutions (50 mg mL-1) revealed that the increased glycopolymer concentrations generally decreased the mAb stability, as judged by the amount of mAb1 ‘monomer’ molecules in solution, with star- and linear-trehalose glycopolymers further generating visible aggregates. Interestingly the latter effect could not have been predicted from the Tm or Tagg experiments conducted at a low concentration regime. Taken together, the data demonstrate a complex interplay of sugar chemistry and molecular topology of the synthetic glycopolymers on their modulation of protein conformational stability and aggregation propensity. Solution concentration was also an important parameter contributing to the stability modulation, and suggests that the stabilising properties of a sugar as the mono- or di-saccharide cannot be extrapolated to the corresponding glycopolymers

Etude de l'action bioprotectrice des sucres : une investigation par dynamique moléculaire et spectroscopie Raman

Understanding the enhanced efficiency of trehalose to stabilise biologicalmolecules compared to other excipients is a matter of intense research. Wehave studied the influence of trehalose and two other stereoisomers, maltoseand sucrose, on the structure and on the dynamics of water and of a modelglobular protein, lysozyme. We have shown that trehalose induces a greaterdestructuration of the water hydrogen bond (HB) network than maltose andsucrose, beyond a concentration of 40-50 wt %, at which the HB network ofsugars percolates. Moreover, the hydration number of sugars and the number ofsugar-sugar HBs suggest that trehalose aqueous solutions are more "homogeneous", for concentrations between 33 and 66 wt %. We have alsolocalised interaction zones between sugars and lysozyme and shown withRaman spectroscopy that trehalose stabilises it more.La compréhension de la plus grande stabilisation des molécules biologiques par un disaccharidetel que le tréhalose par rapport à d'autres excipients fait l'objet de recherches accrues. Nousavons étudié l'influence du tréhalose et de deux autres stéréoisomères, le maltose et le sucrose,sur la structure et sur la dynamique de l'eau et d'une protéine globulaire modèle, le lysozyme. Nousavons montré que le tréhalose induit une plus grande déstructuration du réseau de liaisons hydrogène (LHs)de l'eau que le maltose et le sucrose, au-delà d'une concentration de 40-50 %, à laquelle le réseaude LHs des sucres percole. En outre, le nombre d'hydratation des sucres et lenombre de LHs sucre-sucre suggèrent que les solutions aqueuses de tréhalose sont plus "homogènes",pour des concentrations entre 33 et 66 % pds. Nous avons également localisé les zonesd'interaction des sucres avec le lysozyme et montré par spectroscopie Raman que le tréhalose le stabilisedavantage

How Accurate Is the Egg-Box Model in Describing the Binding of Calcium to Polygalacturonate? A Molecular Dynamics Simulation Study

International audienceWe performed molecular dynamics (MD) simulations of octameric galacturonate, GalA8, chains in the presence of Ca2+ in a ratio of R = [Ca2+]/[GalA] = 0.25 in order to determine to which extent the popular "egg-box model" (EBM) is able to describe the association between Ca2+ cations and polygalacturonate (polyGalA) chains. To this aim, we slig h t l y revised the empirical parameters for the interaction between Ca2+ and the carboxylate oxygen atoms of GalA units so as to reproduce the experimental Ca2+-GalA association constant. We also defined an ad hoc order parameter, referred to as the egg-box score (EBS), that quantifies any deviation of the local coordination geometry of calcium cations with respect to an "ideal" EBM coordination geometry. The results reveal that the local coordination geometry of Ca2+ cations bound to polyGalA chains differs from that of the EBM. Moreover, polyGalA chains exhibit significant conformational disorder, and the cross-l i n k angles formed between polyGalA chain axes are broadly distributed . Overall, the present study suggests that the EBM fails to describe accurately the association modes between calcium and polyGalA chains at a molar ratio R of 0.25

ROLE OF RESIDUAL WATER FOR THE STABILITY OF FREEZE-DRIED PROTEINS

International audienc