Site‐directed analysis on protein hydrophobicity

Hydrophobicity of a protein is considered to be one of the major intrinsic factors dictating the protein aggregation propensity. Understanding how protein hydrophobicity is determined is, therefore, of central importance in preventing protein aggregation diseases and in the biotechnological production of human therapeutics. Traditionally, protein hydrophobicity is estimated based on hydrophobicity scales determined for individual free amino acids, assuming that those scales are unaltered when amino acids are embedded in a protein. Here, we investigate how the hydrophobicity of constituent amino acid residues depends on the protein context. To this end, we analyze the hydration free energy—free energy change on hydration quantifying the hydrophobicity—of the wild‐type and 21 mutants of amyloid‐beta protein associated with Alzheimer's disease by performing molecular dynamics simulations and integral‐equation calculations. From detailed analysis of mutation effects on the protein hydrophobicity, we elucidate how the protein global factor such as the total charge as well as underlying protein conformations influence the hydrophobicity of amino acid residues. Our results provide a unique insight into the protein hydrophobicity for rationalizing and predicting the protein aggregation propensity on mutation, and open a new avenue to design aggregation‐resistant proteins as biotherapeutics. © 2014 Wiley Periodicals, Inc.     

Effect of single-point sequence alterations on the aggregation propensity of a model protein

Sequences of contemporary proteins are believed to have evolved through a process that optimized their overall fitness, including their resistance to deleterious aggregation. Biotechnological processing may expose therapeutic proteins to conditions that are much more conducive to aggregation than those encountered in a cellular environment. An important task of protein engineering is to identify alternative sequences that would protect proteins when processed at high concentrations without altering their native structure associated with specific biological function. Our computational studies exploit parallel tempering simulations of coarse-grained model proteins to demonstrate that isolated amino acid residue substitutions can result in significant changes in the aggregation resistance of the protein in a crowded environment while retaining protein structure in isolation. A thermodynamic analysis of protein clusters subject to competing processes of folding and association shows that moderate mutations can produce effects similar to those caused by changes in system conditions, including temperature, concentration, and solvent composition, that affect the aggregation propensity. The range of conditions where a protein can resist aggregation can therefore be tuned by sequence alterations, although the protein generally may retain its generic ability for aggregation.     

Solvent-exposed backbone loosens the hydration shell of soluble folded proteins

The hydration shell of a soluble folded protein is not uniform: its tightness, marked by the mobility of interfacial water, is site dependent and modulates the propensity for protein associations. We found that the most pronounced interfacial dehydration propensity for representative folds is promoted by solvent-exposed intramolecular hydrogen bonds that are incompletely shielded from water attack. These bonds are poorly wrapped by surrounding nonpolar groups from the side chains and their dehydration is energetically favored.     

Dynamical changes of hemoglobin and its surrounding water during thermal denaturation as studied by quasielastic neutron scattering and temperature modulated differential scanning calorimetry

We have investigated the dynamical behavior of both the protein hemoglobin and its surrounding water during the denaturation process using modulated temperature differential scanning calorimetry, quasielastic neutron scattering, and frequency dependent conductivity measurements. To distinguish between the scattering from the protein and its surrounding water, neutron scattering measurements were performed on both a fully hydrogenated sample as well as a sample where the water and the exchangeable hydrogen atoms on the protein surface were deuterated. The experimental data show that the unfolding and aggregation processes are substantially overlapping in temperature. The unfolding process occurs in the approximate temperature range of 315-345 K, whereas the aggregation process starts around 330-335 K and is completed at 360 K. Furthermore, the results suggest that the secondary structure of the protein unfolds at about 325 K, and that this leads to an increased number of water molecule hydrogen bonded to the protein. Thus, the unfolding of the secondary structure reduces the number of mobile (on the experimental time scale of about 50-100 ps) water molecules. In contrast, the aggregation of protein molecules seems to have a minor effect on the dynamics of its surrounding water. In the case of the protein dynamics there are competing effects from unfolding and aggregation. The unfolding process increases the flexibility of the protein, whereas the initial aggregation reduces its dynamics. The conductivity seems to be negatively affected by both reduced water mobility and an aggregation of protein molecules.     

Characterizing amyloid‐beta protein misfolding from molecular dynamics simulations with explicit water

Extracellular deposition of amyloid‐beta (Aβ) protein, a fragment of membrane glycoprotein called β‐amyloid precursor transmembrane protein (βAPP), is the major characteristic for the Alzheimer's disease (AD). However, the structural and mechanistic information of forming Aβ protein aggregates in a lag phase in cell exterior has been still limited. Here, we have performed multiple all‐atom molecular dynamics simulations for physiological 42‐residue amyloid‐beta protein (Aβ42) in explicit water to characterize most plausible aggregation‐prone structure (APS) for the monomer and the very early conformational transitions for Aβ42 protein misfolding process in a lag phase. Monitoring the early sequential conformational transitions of Aβ42 misfolding in water, the APS for Aβ42 monomer is characterized by the observed correlation between the nonlocal backbone H‐bond formation and the hydrophobic side‐chain exposure. Characteristics on the nature of the APS of Aβ42 allow us to provide new insight into the higher aggregation propensity of Aβ42 over Aβ40, which is in agreement with the experiments. On the basis of the structural features of APS, we propose a plausible aggregation mechanism from APS of Aβ42 to form fibril. The structural and mechanistic observations based on these simulations agree with the recent NMR experiments and provide the driving force and structural origin for the Aβ42 aggregation process to cause AD. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2011     

Atomic decomposition of the protein solvation free energy and its application to amyloid-beta protein in water

We report the development of an atomic decomposition method of the protein solvation free energy in water, which ascribes global change in the solvation free energy to local changes in protein conformation as well as in hydration structure. So far, empirical decomposition analyses based on simple continuum solvation models have prevailed in the study of protein-protein interactions, protein-ligand interactions, as well as in developing scoring functions for computer-aided drug design. However, the use of continuum solvation model suffers serious drawbacks since it yields the protein free energy landscape which is quite different from that of the explicit solvent model and since it does not properly account for the non-polar hydrophobic effects which play a crucial role in biological processes in water. Herein, we develop an exact and general decomposition method of the solvation free energy that overcomes these hindrances. We then apply this method to elucidate the molecular origin for the solvation free energy change upon the conformational transitions of 42-residue amyloid-beta protein (Aβ42) in water, whose aggregation has been implicated as a primary cause of Alzheimer's disease. We address why Aβ42 protein exhibits a great propensity to aggregate when transferred from organic phase to aqueous phase.     

Non‐Equilibrium Polymerization of Cross‐β Amyloid Peptides for Temporal Control of Electronic Properties

Hydrophobic collapse plays crucial roles in protein functions, from accessing the complex three‐dimensional structures of native enzymes to the dynamic polymerization of non‐equilibrium microtubules. However, hydrophobic collapse can also lead to the thermodynamically downhill aggregation of aberrant proteins, which has interestingly led to the development of a unique class of soft nanomaterials. There remain critical gaps in the understanding of the mechanisms of how hydrophobic collapse can regulate such aggregation. Demonstrated herein is a methodology for non‐equilibrium amyloid polymerization through mutations of the core sequence of Aβ peptides by a thermodynamically activated moiety. An out of equilibrium state is realized because of the negative feedback from the transiently formed cross‐β amyloid networks. Such non‐equilibrium amyloid nanostructures were utilized to access temporal control over its electronic properties.     

Electrochemiluminescence Platforms Based on Small Water‐Insoluble Organic Molecules for Ultrasensitive Aqueous‐Phase Detection

Highly efficient detection in the aqueous phase for water‐insoluble organic molecule probes is challenging. The bright aggregated‐state electrochemiluminescence (ECL) of 1,1‐disubstituted 2,3,4,5‐tetraphenylsiloles by a co‐reactant approach was discovered, and a heterogeneous aggregation‐induced emission ECL (HAIE‐ECL) was constructed at the electrode surface, showing very high ECL efficiency (37.8 %) and selective recognition for industrially important DNBP plasticizer with a low detection limit of 0.15 nm in the water phase. A mechanistic study indicates that ECL is mainly generated due to the high electron affinity of siloles and restriction of the intramolecular motions caused by their propeller‐like noncoplanar structures. This system realizes the sensing of organic‐based ECL in the water phase by solving the crucial problems of water insolubility and aggregation‐caused quenching (ACQ), and demonstrates potential for further application because of its design and high efficiency.     

Classification of water molecules in protein binding sites

Water molecules play a crucial role in mediating the interaction between a ligand and a macromolecular receptor. An understanding of the nature and role of each water molecule in the active site of a protein could greatly increase the efficiency of rational drug design approaches: if the propensity of a water molecule for displacement can be determined, then synthetic effort may be most profitably applied to the design of specific ligands with the displacement of this water molecule in mind. In this paper, a thermodynamic analysis of water molecules in the binding sites of six proteins, each complexed with a number of inhibitors, is presented. Two classes of water molecules were identified: those conserved and not displaced by any of the ligands, and those that are displaced by some ligands. The absolute binding free energies of 54 water molecules were calculated using the double decoupling method, with replica exchange thermodynamic integration in Monte Carlo simulations. It was found that conserved water molecules are on average more tightly bound than displaced water molecules. In addition, Bayesian statistics is used to calculate the probability that a particular water molecule may be displaced by an appropriately designed ligand, given the calculated binding free energy of the water molecule. This approach therefore allows the numerical assessment of whether or not a given water molecule should be targeted for displacement as part of a rational drug design strategy.     

Selective Inhibition of Aggregation and Toxicity of a Tau‐Derived Peptide using Its Glycosylated Analogues

Protein glycosylation is a ubiquitous post‐translational modification that regulates the folding and function of many proteins. Misfolding of protein monomers and their toxic aggregation are the hallmark of many prevalent diseases. Thus, understanding the role of glycans in protein aggregation is highly important and could contribute both to unraveling the pathology of protein misfolding diseases as well as providing a means for modifying their course for therapeutic purposes. Using β‐O‐linked glycosylated variants of the highly studied Tau‐derived hexapeptide motif VQIVYK, which served as a simplified amyloid model, we demonstrate that amyloid formation and toxicity can be strongly attenuated by a glycan unit, depending on the nature of the glycan itself. Importantly, we show for the first time that not only do glycans hinder self‐aggregation, but the glycosylated peptides are capable of inhibiting aggregation of the non‐modified corresponding amyloid scaffold.     

A novel preparation method for microspheres by glycerol modified solid‐in‐oil‐in‐water multi‐emulsion

Biodegradable material poly(D, L ‐lactic‐co‐glycolic) acid (PLGA) plays an important role in drug‐sustained release systems. Here, we describe a glycerol modified solid‐in‐oil‐in‐water (m‐S/O/W) emulsion method for PLGA microspheres, in order to encapsulate proteins in PLGA by utilizing dextran glassy particles to protect the proteins from denaturing, unfolding, and aggregation during preparation and new external water phase to prevent the inner dextran glassy particles from leaking into the external water phase. External water phase containing 20, 40, 60, 80% glycerol showed that proteins released faster and more completely with increased glycerol content. According to their varied release profiles, microspheres of different formulations could be used to encapsulate vaccines or for delivering proteins over long‐term. Copyright © 2009 John Wiley & Sons, Ltd.     

Amino acids with hydrogen-bonding side chains have an intrinsic tendency to sample various turn conformations in aqueous solution

Local structure in unfolded proteins, especially turn segments, has been suggested to initiate the hierarchical protein‐folding process. To determine the intrinsic propensity to form such turn structures, amide I′ band profiles of the Raman, IR, and vibrational circular dichroism (VCD) spectra, and several structure‐sensitive NMR J‐coupling constants, have been measured for a series of GxG (x=D, N, T, C) peptides, in which the central x residues are abundant in various turn motifs in folded proteins. In addition, we revisited earlier measured GSG experimental data. To check whether this relatively high propensity for these residues to sample turns reflects an intrinsic propensity, the experimental data were analyzed in terms of conformational distributions that can be described as a superposition of two‐dimensional Gaussian distributions associated with different so‐called mesostates. The analysis reveals that the investigated residues sample dihedral angles similar to those found in the corner residues of various turns, namely, type I/I′, II/II′, and IV β‐turns. Aspartic acid (D) was found to predominantly sample regions attributed to turns, including distributions at the upper border of the upper‐right quadrant of the Ramachandran plot, which bear some resemblance to asx‐turns observed in proteins. This conformation enables hydrogen bonding between the side‐chain carboxylate and the C‐terminal amide group. Altogether, the study shows that the high propensity for T, S, C, N, and D to be located in turn motifs reflects, to a substantial degree, an intrinsic property and supports the role of these residues as initiation sites for hierarchical folding processes that can lead to compact structures in the unfolded state of peptides and proteins.     

The Effect of Surface Charge Saturation on Heat‐induced Aggregation of Firefly Luciferase

We present here the effect of firefly luciferase surface charge saturation and the presence of some additives on its thermal‐induced aggregation. Three mutants of firefly luciferase prepared by introduction of surface Arg residues named as 2R, 3R and 5R have two, three and five additional arginine residues substituted at their surface compared to native luciferase; respectively. Turbidimetric study of heat‐induced aggregation indicates that all three mutants were reproducibly aggregated at higher rates relative to wild type in spite of their higher thermostability. Among them, 2R had most evaluated propensity to heat‐induced aggregation. Therefore, the hydrophilization followed by appearing of more substituted arginine residues with positive charge on the firefly luciferase surface was not reduced its thermal aggregation. Nevertheless, at the same condition in the presence of charged amino acids, e.g. Arg, Lys and Glu, as well as a hydrophobic amino acid, e.g. Val, the heat‐induced aggregation of wild type and mutants of firefly luciferases was markedly decelerated than those in the absence of additives. On the basis of obtained results it seems, relinquishment of variety in charge of amino acid side chains, they via local interactions with proteins cause to decrease rate and extent of their thermal aggregation.     

Confined Water in Amyloid‐Competent Oligomers of the Prion Protein

Conformational switching of the prion protein into the abnormal form involves the formation of (obligatory) molten‐oligomers that mature into ordered amyloid fibrils. The role of water in directing the course of amyloid formation remains poorly understood. Here, we show that the mobility of the water molecules within the on‐pathway oligomers is highly retarded. The water relaxation time within the oligomers was estimated to be ≈1 ns which is about three orders of magnitude slower than the bulk water and resembles the characteristics of (trapped) nano‐confined water. We propose that the coalescence of these obligatory oligomers containing trapped water is entropically favored because of the release of ordered water molecules in the bulk milieu and results in the sequestration of favorable inter‐chain amyloid contacts via nucleated conformational conversion. The dynamic role of water in protein aggregation will have much broader implications in a variety of protein misfolding diseases.     

Temperature‐Responsive Mixed‐Shell Polymeric Micelles for the Refolding of Thermally Denatured Proteins

We have fabricated a mixed‐shell polymeric micelle (MSPM) that closely mimics the natural molecular chaperone GroEL? GroES complex in terms of structure and functionality. This MSPM, which possesses a shared PLA core and a homogeneously mixed PEG and PNIAPM shell, is constructed through the co‐assembly of block copolymers poly(lactide‐b‐poly(ethylene oxide) (PLA‐b‐PEG) and poly(lactide)‐b‐poly(N‐isopropylacryamide) (PLA‐b‐PNIPAM). Above the lower critical solution temperature (LCST) of PNIPAM, the MSPM evolves into a core–shell–corona micelle (CSCM), as a functional state with hydrophobic PNIPAM domains on its surface. Light scattering (LS), TEM, and fluorescence and circular dichroism (CD) spectroscopy were performed to investigate the working mechanism of the chaperone‐like behavior of this system. Unfolded protein intermediates are captured by the hydrophobic PNIPAM domains of the CSCM, which prevent harmful protein aggregation. During cooling, PNIPAM reverts into its hydrophilic state, thereby inducing the release of the bound unfolded proteins. The refolding process of the released proteins is spontaneously accomplished by the presence of PEG in the mixed shell. Carbonic anhydrase B (CAB) was chosen as a model to investigate the refolding efficiency of the released proteins. In the presence of MSPM, almost 93 % CAB activity was recovered during cooling after complete denaturation at 70 °C. Further results reveal that this MSPM also works with a wide spectrum of proteins with more‐complicated structures, including some multimeric proteins. Given the convenience and generality in preventing the thermal aggregation of proteins, this MSPM‐based chaperone might be useful for preventing the toxic aggregation of misfolded proteins in some diseases.     

Targeting S100B with Peptides Encoding Intrinsic Aggregation-Prone Sequence Segments

S100 proteins assume a diversity of oligomeric states including large order self-assemblies, with an impact on protein structure and function. Previous work has uncovered that S100 proteins, including S100B, are prone to undergo β-aggregation under destabilizing conditions. This propensity is encoded in aggregation-prone regions (APR) mainly located in segments at the homodimer interface, and which are therefore mostly shielded from the solvent and from deleterious interactions, under native conditions. As in other systems, this characteristic may be used to develop peptides with pharmacological potential that selectively induce the aggregation of S100B through homotypic interactions with its APRs, resulting in functional inhibition through a loss of function. Here we report initial studies towards this goal. We applied the TANGO algorithm to identify specific APR segments in S100B helix IV and used this information to design and synthesize S100B-derived APR peptides. We then combined fluorescence spectroscopy, transmission electron microscopy, biolayer interferometry, and aggregation kinetics and determined that the synthetic peptides have strong aggregation propensity, interact with S100B, and may promote co-aggregation reactions. In this framework, we discuss the considerable potential of such APR-derived peptides to act pharmacologically over S100B in numerous physiological and pathological conditions, for instance as modifiers of the S100B interactome or as promoters of S100B inactivation by selective aggregation.     

Salt Modulated Fibrillar Aggregation of the Sweet Protein MNEI in Aqueous Solution

The mechanism of conversion of globular native proteins into amyloid fibrils represents one of the most attractive research topics in biophysics, because of its involvement in the development of severe pathologies and in various biotechnological processes. Aqueous medium properties, such as pH and ionic strength, as well as interactions with other species in solution, play a key role in tuning the fibrillization process. Here, we describe a comparative study of the influence of different ions from the Hofmeister series on the thermal unfolding and aggregation propensity of MNEI, a model protein, selected because of its tendency to form amyloid aggregates at acidic pH, even at temperatures well below its melting temperature. By selecting a temperature at which only negligible amounts of protein are unfolded, we have focused on the effect of ions on fibril formation. ThT fluorescence experiments indicated that all the salts examined increased the rate and the extent of fibrillization. Moreover, we found that anions, particularly sulfate, strongly influence the process, which instead is only marginally affected by different cations. Finally, a specific link to the chloride concentration was detected.     

Suppression of Oligomer Formation and Formation of Non‐Toxic Fibrils upon Addition of Mirror‐Image Aβ42 to the Natural l‐Enantiomer

Racemates often have lower solubility than enantiopure compounds, and the mixing of enantiomers can enhance the aggregation propensity of peptides. Amyloid beta (Aβ) 42 is an aggregation‐prone peptide that is believed to play a key role in Alzheimer's disease. Soluble Aβ42 aggregation intermediates (oligomers) have emerged as being particularly neurotoxic. We hypothesized that the addition of mirror‐image d ‐Aβ42 should reduce the concentration of toxic oligomers formed from natural l ‐Aβ42. We synthesized l ‐ and D ‐Aβ42 and found their equimolar mixing to lead to accelerated fibril formation. Confocal microscopy with fluorescently labeled analogues of the enantiomers showed their colocalization in racemic fibrils. Owing to the enhanced fibril formation propensity, racemic Aβ42 was less prone to form soluble oligomers. This resulted in the protection of cells from the toxicity of l ‐Aβ42 at concentrations up to 50 μm . The mixing of Aβ42 enantiomers thus accelerates the formation of non‐toxic fibrils.     

Modification of a Small β‐Barrel Protein,To Give Pseudo‐Amyloid Structures,Inhibits Amyloid β‐Peptide Aggregation

Aggregation of amyloid β‐peptide (Aβ) is closely related to the pathogenesis of Alzheimer’s disease (AD). Although much effort has been devoted to the construction of molecules that inhibit the aggregation of Aβ1‐42, high doses are needed for the inhibition of Aβ aggregation in many cases. Previously, we reported that designed green fluorescent protein (GFP) analogues that gives pseudo‐Aβ β‐sheet structures can work as an aggregation inhibitor against Aβ. To further test this design strategy, we constructed protein analogues that mimic Aβ β‐sheet structures of amyloids by using insulin‐like growth factor 2 receptor domain 11 (IGF2R‐d11) as a scaffold. A designed protein, named IG11KK, which has a parallel configuration of Aβ‐like β sheets, can bind more preferentially to oligomeric Aβ1‐42 than the monomer. Moreover, IG11KK suppressed the aggregation of Aβ1‐42 efficiently, even though lower concentrations of IG11KK than Aβ were used. The aggregation kinetics of Aβ in the presence of the designed proteins revealed that IG11KK can work as an inhibitor not only for the early to middle stages, but also in the latter stage of Aβ aggregation owing to its favorable binding to oligomeric structures of Aβ. The design strategy using β‐barrel proteins such as IGF2R‐d11 and GFP is useful in generating excellent inhibitors of protein misfolding and amyloid formation.