Effects of arginine on heat-induced aggregation of concentrated protein solutions

Arginine is one of the commonly used additives to enhance refolding yield of proteins, to suppress aggregation of proteins, and to increase solubility of proteins, and yet the molecular interactions that contribute to the role of arginine are unclear. Here, we present experiments, using bovine serum albumin (BSA), lysozyme (LYZ), and β-lactoglobulin (BLG) as model proteins, to show that arginine can enhance heat-induced aggregation of concentrated protein solutions, contrary to the conventional belief that arginine is a universal suppressor of aggregation. Results show that the enhancement in aggregation is caused only for BSA and BLG, but not for LYZ, indicating that arginine's preferential interactions with certain residues over others could determine the effect of the additive on aggregation. We use this previously unrecognized behavior of arginine, in combination with density functional theory calculations, to identify the molecular-level interactions of arginine with various residues that determine arginine's role as an enhancer or suppressor of aggregation of proteins. The experimental and computational results suggest that the guanidinium group of arginine promotes aggregation through the hydrogen-bond-based bridging interactions with the acidic residues of a protein, whereas the binding of the guanidinium group to aromatic residues (aggregation-prone) contributes to the stability and solubilization of the proteins. The approach, we describe here, can be used to select suitable additives to stabilize a protein solution at high concentrations based on an analysis of the amino acid content of the protein.     

Non-native protein aggregation kinetics

Experimental kinetics of non-native protein aggregation are of practical importance in that they help dictate viable processing, formulation, and storage conditions for biotechnology products, and appear to play a role in determining the onset of a number of diseases. Fundamentally, aggregation kinetics provide insights into the identity of key intermediates in the process, and quantitative tests of available models of aggregation. Although aggregation kinetics often display seemingly disparate behaviors across different proteins and sample conditions, this review illustrates how many of these can be understood within a general framework that treats aggregation as a multi-stage process, and how most available kinetic models of aggregation can be grouped hierarchically in terms of which stage(s) they include. This provides an aid for workers seeking a mechanistic interpretation of in vitro aggregation kinetics, for discriminating among competing models, and in designing experiments to assess in vitro protein stability. Limitations and the utility of purely kinetic approaches to studying aggregation, clarifications of common misperceptions regarding experimental aggregation kinetics, and some outstanding challenges in the field are briefly discussed.     

Computational approaches to understanding protein aggregation in neurodegeneration

The generation of toxic non-native protein conformers has emerged as a unifying thread among disorders such as Alzheimer＇s disease, Parkinson＇s disease, and amyotrophic lateral sclerosis. Atomic-level detail regarding dynamical changes that facilitate protein aggre- gation, as well as the structural features of large-scale ordered aggregates and soluble non-native oligomers, would contribute signifi- cantly to current understanding of these complex phenomena and offer potential strategies for inhibiting formation of cytotoxic species. However, experimental limitations often preclude the acquisition of high-resolution structural and mechanistic information for aggregating systems. Computational methods, particularly those combine both aU-atom and coarse-grained simulations to cover a wide range of time and length scales, have thus emerged as crucial tools for investigating protein aggregation. Here we review the current state of computational methodology for the study of protein self-assembly, with a focus on the application of these methods toward understanding of protein aggregates in human neurodegenerative disorders.     

Insight into the correlation between lag time and aggregation rate in the kinetics of protein aggregation

Under favorable conditions, many proteins can assemble into macroscopically large aggregates such as the amyloid fibrils that are associated with Alzheimer's, Parkinson's, and other neurological and systemic diseases. The overall process of protein aggregation is characterized by initial lag time during which no detectable aggregation occurs in the solution and by maximal aggregation rate at which the dissolved protein converts into aggregates. In this study, the correlation between the lag time and the maximal rate of protein aggregation is analyzed. It is found that the product of these two quantities depends on a single numerical parameter, the kinetic index of the curve quantifying the time evolution of the fraction of protein aggregated. As this index depends relatively little on the conditions and/or system studied, our finding provides insight into why for many experiments the values of the product of the lag time and the maximal aggregation rate are often equal or quite close to each other. It is shown how the kinetic index is related to a basic kinetic parameter of a recently proposed theory of protein aggregation. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Interplay between protein homeostasis networks in protein aggregation and proteotoxicity

The misfolding and aggregation of disease proteins is characteristic of numerous neurodegenerative diseases. Particular neuronal populations are more vulnerable to proteotoxicity while others are more apt to tolerate the misfolding and aggregation of disease proteins. Thus, the cellular environment must play a significant role in determining whether disease proteins are converted into toxic or benign forms. The endomembrane network of eukaryotes divides the cell into different subcellular compartments that possess distinct sets of molecular chaperones and protein interaction networks. Chaperones act as agonists and antagonists of disease protein aggregation to prevent the accumulation of toxic intermediates in the aggregation pathway. Interacting partners can also modulate the conformation and localization of disease proteins and thereby influence proteotoxicity. Thus, interplay between these protein homeostasis network components can modulate the self‐association of disease proteins and determine whether they elicit a toxic or benign outcome. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 229–236, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Intracellular distribution of heat-induced stress glycoproteins

Cellular heat stress results in elevated heat-shock protein (HSP) synthesis and in thermotolerance development. Recently, we demonstrated that protein glycosylation is also an integral part of the stress response with the identification of two major stress glycoproteins, GP50, associated with thermotolerance, and P-SG67, the “prompt” stress glycoprotein induced immediately during acute heat stress. In the present study, we characterized the subcellular location and redistribution of these proteins during the cellular injury and recovery phase. In unheated and heated CHO cells, both stress glycoproteins were present in each subcellular fraction isolated by differential centrifugation. However, the subcellular redistribution in the course of cellular recovery after heat stress was specific for each stress glycoprotein. GP50 was present in all subcellular fractions before heat stress, but showed relatively little redistribution after heat stress. By 24 h of recovery following stress, GP50 showed partial depletion from lysosomes and microsomes, and was mainly present in the mitochondria. Glycosylated P-SG67 was redistributed in a more complex fashion. It was seen predominantly in the lysosomes and microsomes immediately following heat-stress, but after 6 h of recovery following heat stress, it largely disappeared from the microsomes and was present mainly in the cytosol. By 24 h of recovery following heat stress, it was found predominantly in the nucleus-rich fraction and mitochondria. The localization of GP50 and P-SG67 by subcellular fractionation is consistent with immunolocalization studies and contrasts with the translocation of HSP70 after heat stress from cytosol to nuclei and nucleoli. These results reflect a characteristic distribution for each stress glycoprotein; their presence in virtually all subcellular fractions suggests multifunctional roles for the various stress glycoproteins in the cellular heat stress response. J. Cell. Biochem. 66:98–111, 1997. © 1997 Wiley-Liss, Inc.     

The addition of SDS to the Bradford dye-binding protein assay,a modification with increased sensitivity to collagen

The standard Bradford protein assay is insensitive to collagen. But if a small, sub-threshold amount of SDS is added to the sample, the response to collagen in increased by at least an order of magnitude, while, on average, the sensitivity for non-collagens is decreased by approximately a factor of 2. As a result comparable color formation is achieved with both collagens and non-collagens. The addition of protein to a sub-threshold amount of SDS results in the formation of a green color measurable as an increase in absorbance at 700 nm, in contrast to the blue color measured at 595 nm in the standard assay. Depending upon the source, the threshold level for SDS varies from 30 to 50 μg. The response to protein is linear up to approximately 40 μg of protein per ml of reagent.     

Neurofilament protein aggregation in a cell line model system

Protein aggregates are associated with many diseases and even aggregates of proteins that have no role in disease are inherently toxic to both neuronal and non-neuronal cells. We have developed a model system to explore the mechanism of protein aggregation using a mouse muscle cell line expressing chimeric neurofilament (NF) proteins, a constituent of the protein aggregates in ALS, Lewy body dementia, and Charcot-Marie-Tooth disease. Formation of protein aggregates in these cells leads to reduced cell viability and activated caspases. Aggregates contained both chimeric NF proteins and ubiquitin by immunolocalization and were predominately cytosolic when proteins were expressed at low levels or for shorter periods of time but were present in the nucleus when expression levels increased. This system represents a flexible, new tool to decipher the molecular mechanism of protein aggregation and the contributions of aggregation to cell toxicity.     

Aggregated proteins accelerate but do not increase the aggregation of D-glyceraldehyde-3-phosphate dehydrogenase. Specificity of protein aggregation

The effect of protein aggregates on the aggregation of d-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during unfolding and refolding has been studied. The aggregation of GAPDH follows a sigmoid course. The presence of protein aggregates increases the aggregation rate during unfolding and refolding of GAPDH but does not change the extent of aggregation and the final renaturation yield. It is suggested that protein aggregates function as seeds for aggregation via hydrophobic interaction with only GAPDH folding intermediates destined to aggregate and do not affect the distribution between pathways leading to correct folding and aggregation. Moreover, two different proteins do not interfere with each other during their simultaneous refolding together in a buffer. These findings provide insight into a mechanism by which cells prevent protein folding against the interference from aggregation of other proteins.     

Proline inhibits aggregation during protein refolding

The in vitro refolding of hen egg-white lysozyme is studied in the presence of various osmolytes. Proline is found to prevent aggregation during protein refolding. However, other osmolytes used in this study fail to exhibit a similar property. Experimental evidence suggests that proline inhibits protein aggregation by binding to folding intermediate(s) and trapping the folding intermediate(s) into enzymatically inactive, "aggregation-insensitive" state(s). However, elimination of proline from the refolded protein mixture results in significant recovery of the bacteriolytic activity. At higher concentrations (>1.5 M), proline is shown to form loose, higher-order molecular aggregate(s). The supramolecular assembly of proline is found to possess an amphipathic character. Formation of higher-order aggregates is believed to be crucial for proline to function as a protein folding aid. In addition to its role in osmoregulation under water stress conditions, the results of this study hint at the possibility of proline behaving as a protein folding chaperone.     

Protein aggregation determinants from a simplified model: cooperative folders resist aggregation

Two-chain aggregation simulations using minimalist models of proteins G, L, and mutants were used to investigate the fundamentals of protein aggregation. Mutations were selected to break up repeats of hydrophobic beads in the sequence while maintaining native topology and folding ability. Data are collected under conditions in which all chain types have similar folded populations and after equilibrating the separated chains to minimize competition between folding and aggregation. Folding cooperativity stands out as the best single-chain determinant under these conditions and for these simple models. It can be experimentally measured by the width of the unfolding transition during thermal denaturation and loosely related to population of intermediate-like states during folding. Additional measures of cooperativity and other properties such as radius of gyration fluctuations and patterning of hydrophobic residues are also examined. Initial contact system states with transition-state characteristics can be identified and are more expanded than average initial contact states. Two-chain minimalist model aggregates are considerably less structured than their native states and have minimal domain-swapping features.     

Modulation of protein aggregation by polyethylene glycol conjugation: GCSF as a case study

Polyethylene glycol (PEG) conjugation to proteins has emerged as an important technology to produce drug molecules with sustained duration in the body. However, the implications of PEG conjugation to protein aggregation have not been well understood. In this study, conducted under physiological pH and temperature, N-terminal attachment of a 20 kDa PEG moiety to GCSF had the ability to (1) prevent protein precipitation by rendering the aggregates soluble, and (2) slow the rate of aggregation relative to GCSF. Our data suggest that PEG-GCSF solubility was mediated by favorable solvation of water molecules around the PEG group. PEG-GCSF appeared to aggregate on the same pathway as that of GCSF, as evidenced by (a) almost identical secondary structural transitions accompanying aggregation, (b) almost identical covalent character in the aggregates, and (c) the ability of PEG-GCSF to rescue GCSF precipitation. To understand the role of PEG length, the aggregation properties of free GCSF were compared to 5kPEG-GCSF and 20kPEG-GCSF. It was observed that even 5kPEG-GCSF avoided precipitation by forming soluble aggregates, and the stability toward aggregation was vastly improved compared to GCSF, but only marginally less stable than the 20kPEG-GCSF. Biological activity measurements demonstrated that both 5kPEG-GCSF and 20kPEG-GCSF retained greater activity after incubation at physiological conditions than free GCSF, consistent with the stability measurements. The data is most compatible with a model where PEG conjugation preserves the mechanism underlying protein aggregation in GCSF, steric hindrance by PEG influences aggregation rate, while aqueous solubility is mediated by polar PEG groups on the aggregate surface.     

Control of aggregation in protein refolding: a variety of surfactants promote renaturation of carbonic anhydrase II.

The denaturation and renaturation of carbonic anhydrase II (CAII) has been studied in several laboratories. Both thermodynamic and kinetic evidence support the existence of at least two intermediates between denatured and native protein. Previous studies have shown that on rapid dilution of a CAII solution from 5 M to 1 M guanidinium chloride, aggregation strongly competes with renaturation at higher protein concentrations, suggesting an upper limit for [CAII] of approximately 0.1%. Our experiments show 60% renaturation at 0.4% [CAII] and that aggregate formation is partially reversible. This yield can be substantially increased by several surfactant additives, including simple alkanols as well as micelle-forming surfactants. Effective surfactants (promoters) act by suppressing initial aggregate formation, not by dissolving aggregates. Promoters act on either the first folding intermediate (I1) or oligomers thereof. Eight of the 18 surfactants examined showed promoter activity, and no correlation was evident between promoter activity and chemical structure or surface tension lowering. These results indicate discrimination (molecular recognition) by I1 and/or its oligomers.     

Effect of denaturant and protein concentrations upon protein refolding and aggregation: a simple lattice model.

We present a study of the competition between protein refolding and aggregation for simple lattice model proteins. The effect of solvent conditions (i.e., the denaturant concentration and the protein concentration) on the folding and aggregation behavior of a system of simple, two-dimensional lattice protein molecules has been investigated via (dynamic Monte Carlo simulations. The population profiles and aggregation propensities of the nine most populated intermediate configurations exhibit a complex dependence on the solution conditions that can be understood by considering the competition between intra- and interchain interactions. Some of these configurations are not even seen in isolated chain simulations; they are observed to be highly aggregation prone and are stabilized primarily by the aggregation reaction in multiple-chain systems. Aggregation arises from the association of partially folded intermediates rather than from the association of denatured random-coil states. The aggregation reaction dominates over the folding reaction at high protein concentration and low denaturant concentration, resulting in low refolding yields at those conditions. However, optimum folding conditions exist at which the refolding yield is a maximum, in agreement with some experimental observations.     

Translation‐coupled protein folding assay using a protease to monitor the folding status

Protein folding is an essential prerequisite for proteins to execute nearly all cellular functions. There is a growing demand for a simple and robust method to investigate protein folding on a large‐scale under the same conditions. We previously developed a global folding assay system, in which proteins translated using an Escherichia coli‐based cell‐free translation system are centrifuged to quantitate the supernatant fractions. Although the assay is based on the assumption that the supernatants contain the folded native states, the supernatants also include nonnative unstructured proteins. In general, proteases recognize and degrade unstructured proteins, and thus we used a protease to digest the unstructured regions to monitor the folding status. The addition of Lon protease during the translation of proteins unmasked subfractions, not only in the soluble fractions but also in the aggregation‐prone fractions. We translated ～90 E. coli proteins in the protease‐inclusion assay, in the absence and presence of chaperones. The folding assay, which sheds light on the molecular mechanisms underlying the aggregate formation and the chaperone effects, can be applied to a large‐scale analysis.     

The competition between protein folding and aggregation: off-lattice minimalist model studies

Protein aggregation has been associated with a number of human diseases, and is a serious problem in the manufacture of recombinant proteins. Of particular interest to the biotechnology industry is deleterious aggregation that occurs during the refolding of proteins from inclusion bodies. As a complement to experimental efforts, computer simulations of multi-chain systems have emerged as a powerful tool to investigate the competition between folding and aggregation. Here we report results from Langevin dynamics simulations of minimalist model proteins. Order parameters are developed to follow both folding and aggregation. By mapping natural units to real units, the simulations are shown to be carried out under experimentally relevant conditions. Data pertaining to the contacts formed during the association process show that multiple mechanisms for aggregation exist, but certain pathways are statistically preferred. Kinetic data show that there are multiple time scales for aggregation, although most association events take place at times much shorter than those required for folding. Last, we discuss results presented here as a basis for future work aimed at rational design of mutations to reduce aggregation propensity, as well as for development of small-molecular weight refolding enhancers.     

Minimalist protein model as a diagnostic tool for misfolding and aggregation

We propose a realistic coarse-grained protein model and a technique to "anchor" the model to available experimental data. We apply this procedure to characterize the effect of multiple mutations on the folding mechanism of protein S6. We show that the mutation of a few "gatekeeper" residues triggers significant changes on the folding landscape of S6. These results suggest that gatekeeper residues control the flexibility of critical regions of S6, that in turn regulates the delicate balance between folding and aggregation. Although obtained with a minimalist protein model, these results are fully consistent with experimental evidence and offer a clue to understand the interplay between folding and aggregation in protein S6.     

Weak protein interactions and pH- and temperature-dependent aggregation of human Fc1

The Fc (fragment crystallizable) is a common structural region in immunoglobulin gamma (IgG) proteins, IgG-based multi-specific platforms, and Fc-fusion platform technologies. Changes in conformational stability, protein-protein interactions, and aggregation of NS0-produced human Fc1 were quantified experimentally as a function of pH (4 to 6) and temperature (30 to 77°C), using a combination of differential scanning calorimetry, laser light scattering, size-exclusion chromatography, and capillary electrophoresis. The Fc1 was O-glycosylated at position 3 (threonine), and confirmed to correspond to the intact IgG1 by comparison with Fc1 produced by cleavage of the parent IgG1. Changing the pH caused large effects for thermal unfolding transitions, but it caused surprisingly smaller effects for electrostatic protein-protein interactions. The aggregation behavior was qualitatively similar across different solution conditions, with soluble dimers and larger oligomers formed in most cases. Aggregation rates spanned approximately 5 orders of magnitude and could be divided into 2 regimes: (i) Arrhenius, unfolding-limited aggregation at temperatures near or above the midpoint-unfolding temperature of the C_H2 domain; (ii) a non-Arrhenius regime at lower temperatures, presumably as a result of the temperature dependence of the unfolding enthalpy for the C_H2 domain. The non-Arrhenius regime was most pronounced for lower temperatures. Together with the weak protein-protein repulsions, these highlight challenges that are expected for maintaining long-term stability of biotechnology products that are based on human Fc constructs.