Quantifying the Reversible Association of Thermosensitive Nanoparticles

Under many conditions, biomolecules and nanoparticles associate by means of attractive bonds, due to hydrophobic attraction. Extracting the microscopic association or dissociation rates from experimental data is complicated by the dissociation events and by the sensitivity of the binding force to temperature (T). Here we introduce a theoretical model that combined with light-scattering experiments allows us to quantify these rates and the reversible binding energy as a function of T. We apply this method to the reversible aggregation of thermoresponsive polystyrene/poly(N-isopropylacrylamide) core-shell nanoparticles, as a model system for biomolecules. We find that the binding energy changes sharply with T, and relate this remarkable switchable behavior to the hydrophobic-hydrophilic transition of the thermosensitive nanoparticles

Dynamics of Structural Elements of GB1 β-Hairpin Revealed by Tryptophan-Cysteine Contact Formation Experiments

Quenching of the triplet state of tryptophan by close contact with cysteine provides a tool for measuring the rate of intramolecular contact formation, one of the most elementary events in the folding process, in peptides and proteins using only natural probes. Here we present a study performed on a stabilized mutant of the second \u3b2-hairpin of the GB1 domain, where we combine steady-state fluorescence, laser-induced temperature-jump, and contact formation measurements to unveil the role of elementary structural components on hairpin dynamics and overall stability. In particular, our methodology provides access to the conformational dynamics of both the folded and unfolded state of the hairpin under native conditions, revealing the presence of extremely slow dynamics on the microsecond time scale in the unfolded state and coexistence of structures with partial pairing of the tails in the folded state. Comparing model peptides that mimic the turn sequence, we found that both ion pairing and hydrogen bonding due to the threonine side chain contribute to the propensity of turn formation but not to the much slower dynamics of the hydrophobic core formation. Interestingly, the dynamics of the turn region in isolation are significantly faster than the dynamics measured for the unfolded state of the complete hairpin, suggesting that non-native hydrophobic contacts slow down the reconfiguration dynamics of the unfolded state. Overall, the information extracted from these experiments provides kinetic limits on interconversions among conformational populations, hence enabling a simplified multistate free-energy landscape for the GB1 hairpin to be drawn

A Protein Aggregation Based Test for Screening of the Agents Affecting Thermostability of Proteins

To search for agents affecting thermal stability of proteins, a test based on the registration of protein aggregation in the regime of heating with a constant rate was used. The initial parts of the dependences of the light scattering intensity (I) on temperature (T) were analyzed using the following empiric equation: I = Kagg(T−T0)2, where Kagg is the parameter characterizing the initial rate of aggregation and T0 is a temperature at which the initial increase in the light scattering intensity is registered. The aggregation data are interpreted in the frame of the model assuming the formation of the start aggregates at the initial stages of the aggregation process. Parameter T0 corresponds to the moment of the origination of the start aggregates. The applicability of the proposed approach was demonstrated on the examples of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscles and bovine liver glutamate dehydrogenase studied in the presence of agents of different chemical nature. The elaborated approach to the study of protein aggregation may be used for rapid identification of small molecules that interact with protein targets

Extrinsic Fluorescent Dyes as Tools for Protein Characterization

Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation. The main underlying mechanisms, which explain the fluorescence properties of many extrinsic dyes, are solvent relaxation processes and (twisted) intramolecular charge transfer reactions, which are affected by the environment and by interactions of the dyes with proteins. In recent time, the use of extrinsic fluorescent dyes such as ANS, Bis-ANS, Nile Red, Thioflavin T and others has increased, because of their versatility, sensitivity and suitability for high-throughput screening. The intention of this review is to give an overview of available extrinsic dyes, explain their spectral properties, and show illustrative examples of their various applications in protein characterization

The feeding tube of cyst nematodes: characterisation of protein exclusion

Plant parasitic nematodes comprise several groups; the most economically damaging of these are the sedentary endoparasites. Sedentary endoparasitic nematodes are obligate biotrophs and modify host root tissue, using a suite of effector proteins, to create a feeding site that is their sole source of nutrition. They feed by withdrawing host cell assimilate from the feeding site though a structure known as the feeding tube. The function, composition and molecular characteristics of feeding tubes are poorly characterised. It is hypothesised that the feeding tube facilitates uptake of host cell assimilate by acting as a molecular sieve. Several studies, using molecular mass as the sole indicator of protein size, have given contradictory results about the exclusion limits of the cyst nematode feeding tube. In this study we propose a method to predict protein size, based on protein database coordinates in silico. We tested the validity of these predictions using travelling wave ion mobility spectrometry--mass spectrometry, where predictions and measured values were within approximately 6%. We used the predictions, coupled with mass spectrometry, analytical ultracentrifugation and protein electrophoresis, to resolve previous conflicts and define the exclusion characteristics of the cyst nematode feeding tube. Heterogeneity was tested in the liquid, solid and gas phase to provide a comprehensive evaluation of three proteins of particular interest to feeding tube size exclusion, GFP, mRFP and Dual PI. The data and procedures described here could be applied to the design of plant expressed defence compounds intended for uptake into cyst nematodes. We also highlight the need to assess protein heterogeneity when creating novel fusion proteins

Folding Kinetics of Small Proteins Revealed by Tryptophan-Cysteine Contact Formation Experiments

Spectroscopic probes sensitive to the formation intra-chain contacts are increasingly used to study the structural and dynamical properties of polypeptides. Quenching of the triplet state of tryptophan by close contact with cysteine enables the measure of contact formation rates without the need of extrinsic probes, thus being suitable for the study of natural proteins and peptides. We illustrate the use of this method to investigate the conformational dynamics of small two-state proteins and of beta-hairpin peptides in conditions close to native. The coexistence of folded and unfolded states can be revealed from the non-exponential relaxation of the excited triplet, enabling the characterization of both unfolded chain dynamics and folding kinetics. The comparison with protein fragments and model disordered peptides allows estimating the contribution of different chain regions to the folding process. Using this approach, we outline the kinetic pathway leading to the formation of the beta-hairpin structure of the C-terminal fragment of protein GB1, revealing the presence of misfolded states, as proposed in recent computational studies

Protein-folding landscapes in multichain systems

Computational studies of proteins have significantly improved our understanding of protein folding. These studies are normally carried out by using chains in isolation. However, in many systems of practical interest, proteins fold in the presence of other molecules. To obtain insight into folding in such situations, we compare the thermodynamics of folding for a Miyazawa–Jernigan model 64-mer in isolation to results obtained in the presence of additional chains. The melting temperature falls as the chain concentration increases. In multichain systems, free-energy landscapes for folding show an increased preference for misfolded states. Misfolding is accompanied by an increase in interprotein interactions; however, near the folding temperature, the transition from folded chains to misfolded and associated chains is entropically driven. A majority of the most probable interprotein contacts are also native contacts, suggesting that native topology plays a role in early stages of aggregation

Measuring internal friction of an ultrafast-folding protein

Nanosecond laser T-jump was used to measure the viscosity dependence of the folding kinetics of the villin subdomain under conditions where the viscogen has no effect on its equilibrium properties. The dependence of the unfolding/refolding relaxation time on solvent viscosity indicates a major contribution to the dynamics from internal friction. The internal friction increases with increasing temperature, suggesting a shift in the transition state along the reaction coordinate toward the native state with more compact structures, and therefore, a smaller diffusion coefficient due to increased landscape roughness. Fitting the data with an Ising-like model yields a relatively small position dependence for the diffusion coefficient. This finding is consistent with the excellent correlation found between experimental and calculated folding rates based on free energy barrier heights using the same diffusion coefficient for every protein