Lysozyme crystallization rates controlled by anomalous fluctuations

Nucleation of protein aggregates and crystals is a process activated by statistical fluctuations of concentration. Nucleation rates may change by several orders of magnitude upon apparently minor changes in the multidimensional space of parameters (temperature, pH, protein concentration, salt type and concentrations, additives). We use available data on hen egg lysozyme crystal induction times in different solution conditions. We measure by static and dynamic light scattering the amplitudes and lifetimes of anomalously ample and long-lived fluctuations occurring in proximity of the liquid-liquid demixing region of the given lysozyme solutions. This allows determining the related spinodal temperatures TS and ε=(T-TS)/TS. Experimental induction times appear to depend solely upon ε over many orders of magnitude. This is quantitatively accounted for in terms of an extended two-stage nucleation model, which jointly takes into consideration amplitudes, lifetimes and scaling properties of anomalous fluctuations. One and the same relation describes quantitatively and equally well the present case of lysozyme crystallization (the best studied case of protein crystallization) and that of sickle hemoglobin fiber formation (the best studied case of protein fiber formation). Comparison with other recent models shows that taking into account lifetimes of anomalous fluctuations allows capturing the essence of the observed behavior

Solvent-induced free energy landscape and solute-solvent dynamic coupling in a multielement solute

Molecular dynamics simulations using a simple multielement model solute with internal degrees of freedom and accounting for solvent-induced interactions to all orders in explicit water are reported. The potential energy landscape of the solute is flat in vacuo. However, the sole untruncated solvent-induced interactions between apolar (hydrophobic) and charged elements generate a rich landscape of potential of mean force exhibiting typical features of protein landscapes. Despite the simplicity of our solute, the depth of minima in this landscape is not far in size from free energies that stabilize protein conformations. Dynamical coupling between configurational switching of the system and hydration reconfiguration is also elicited. Switching is seen to occur on a time scale two orders of magnitude longer than that of the reconfiguration time of the solute taken alone, or that of the unperturbed solvent. Qualitatively, these results are unaffected by a different choice of the water-water interaction potential. They show that already at an elementary level, solvent-induced interactions alone, when fully accounted for, can be responsible for configurational and dynamical features essential to protein folding and function

Effect of T-R conformational change on Sickle Haemoglobin interactions and aggregation

We compare the role of a conformational switch and that of a point mutation in the thermodynamic stability of a protein solution and in the consequent propensity toward aggregation. We study sickle-cell hemoglobin (HbS), the β6 Glu-Val point mutant of adult human hemoglobin (HbA), in its R (CO-liganded) conformation, and compare its aggregation properties to those of both HbS and HbA in their T (unliganded) conformation. Static and dynamic light scattering measurements performed for various hemoglobin concentrations showed critical divergences with mean field exponents as temperature was increased. This allowed determining spinodal data points TS(c) by extrapolation. These points were fitted to theoretical expressions of the TS(c) spinodal line, which delimits the region where the homogeneous solution becomes thermodynamically unstable against demixing in two sets of denser and dilute mesoscopic domains, while remaining still liquid. Fitting provided model-free numerical values of enthalpy and entropy parameters measuring the stability of solutions against demixing, namely, 93.2 kJ/ mol and 314 J/°K-mol, respectively. Aggregation was observed also for R-HbS, but in amorphous form and above physiological temperatures close to the spinodal, consistent with the role played in nucleation by anomalous fluctuations governed by the parameter ε = (T-TS)/TS. Fourier transform infrared (FTIR) and optical spectroscopy showed that aggregation is neither preceded nor followed by denaturation. Transient multiple interprotein contacts occur in the denser liquid domains for R-HbS, T-HbS, and T-HbA The distinct effects of their specific nature and configurations, and those of desolvation on the demising and aggregation thermodynamics, and on the aggregate structure are highlighted