Enzyme-induced aggregation and gelation of proteins

This paper provides a brief overview of the effects of protein hydrolysis on aggregation and gel forming properties of protein systems. Among the food globular proteins, whey proteins and soy proteins are the most extensively studied for their ability to form different textures upon proteolysis. Recent studies were focused on identifying aggregating peptides and on mechanisms of aggregation and gelation.     

Pathways for protein disulphide bond formation

The folding of many secretory proteins depends upon the formation of disulphide bonds. Recent advances in genetics and cell biology have outlined a core pathway for disulphide bond formation in the endoplasmic reticulum (ER) of eukaryotic cells. In this pathway, oxidizing equivalents flow from the recently identified ER membrane protein Ero1p to secretory proteins via protein disulphide isomerase (PDI). Contrary to prior expectations, oxidation of glutathione in the ER competes with oxidation of protein thiols. Contributions of PDI homologues to the catalysis of oxidative folding will be discussed, as will similarities between eukaryotic and prokaryotic disulphide-bond-forming systems.     

Disulphide bond assignment in human tissue inhibitor of metalloproteinases (TIMP).

Disulphide bonds in human recombinant tissue inhibitor of metalloproteinases (TIMP) were assigned by resolving proteolytic digests of TIMP on reverse-phase h.p.l.c. and sequencing those peaks judged to contain disulphide bonds by virtue of a change in retention time on reduction. This procedure allowed the direct assignment of Cys-145-Cys-166 and the isolation of two other peptides containing two disulphide bonds each. Further peptide cleavage in conjunction with fast-atom-bombardment m.s. analysis permitted the assignments Cys-1-Cys-70, Cys-3-Cys-99, Cys-13-Cys-124 and Cys-127-Cys-174 from these peptides. The sixth bond Cys-132-Cys-137 was assigned by inference, as the native protein has no detectable free thiol groups.     

Interchain disulfide bond formation in types I and II procollagen. Evidence for a protein disulfide isomerase catalyzing bond formation

The assembly of reduced pro-alpha chains of type I and type II procollagen into the native triple-helical molecule was examined in vitro in the presence and absence of pure protein disulfide isomerase. The data clearly indicates that protein disulfide isomerase is able to accelerate the formation of native interchain disulfide bonds in these procollagens. It takes about 6 min after disulfide bonding before triple-helical molecules exist, while the time required to produce triple-helical type I procollagen in the presence of protein disulfide isomerase is 9.4 min and that for type II procollagen 17.2 min. These values agree with those obtained for type I and II procollagen in vivo suggesting that protein disulfide isomerase is also an enzyme catalyzing interchain disulfide bond formation in procollagen in vivo. The formation of native disulfide bonds can proceed without any enzyme catalysis but then requires the presence of reduced and oxidized glutathione. Bonding is rather slow in such a case, however, resulting in a delay in the formation of the triple helix.     

Mechanical characterization of network formation during heat-induced gelation of whey protein dispersions

The formation of gel network structures during isothermal heating of whey protein aqueous dispersions was probed by mechanical spectroscopy. It was anticipated that the pathway of the sol-to-gel transition of whey protein dispersions is quite different from that of ordinary cross-linking polymers (e.g., percolation-type transition), since aqueous solutions of native whey proteins have been shown to be highly structured even before gelation, in our previous study. At 20 degrees C, aqueous dispersions of beta-lactoglobulin, the major whey protein, and those of whey protein isolate (WPI), a mixture of whey proteins, exhibited solid-like mechanical spectra, i.e., the predominant storage modulus G' over the loss modulus G", in a certain range of the frequency omega (1-100 rad/s), regardless of the presence or absence of added NaCl. The existence of the added salt was, however, a critical factor for determining transitions in mechanical spectra during gelation at 70 degrees C. beta-Lactoglobulin dispersions in 0.1 mol/dm(3) NaCl maintained the solid-like nature during the entire gelation process and, after passing through the gelation point, satisfied parallel power laws (G' approximately G" approximately omega(n)) that have been proposed for a critical gel (i.e., the gel at the gelation point) that possesses a self-similar or fractal network structure. In contrast, beta-lactoglobulin dispersions without added salt exhibited a transition from solid-like [G'(omega) > G"(omega)] to liquid-like [G'(omega) < G"(omega)] mechanical spectra before gelation, but no parallel power law behavior was recognized at the gelation point. During extended heating time (aging), beta-lactoglobulin gels with 0.1 mol/dm(3) NaCl showed deviations from the parallel power laws, while spectra of gels without added NaCl approached the parallel power laws, suggesting that post-gelation reactions also significantly affect gel network structures. A percolation-type sol-to-gel transition was found only for WPI dispersions without added salt.     

Inhibition of protein aggregation and amyloid formation by small molecules

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Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy

Protein aggregation generally results from association between hydrophobic regions of individual monomers. However, additional mechanisms arising from specific interactions, such as intermolecular disulfide bond formation, may also contribute to the process. The latter is proposed to be the initiating pathway for aggregation of immunoglobulin (IgG), which is essential for triggering its immune response. To test the veracity of this hypothesis, we have employed fluorescence correlation spectroscopy to measure the kinetics of aggregation of IgG in separate experiments either allowing or inhibiting disulfide formation. Fluorescence correlation spectroscopy measurements yielded a diffusion time (τ_D) of ～200 µsec for Rhodamine‐labeled IgG, corresponding to a hydrodynamic radius (R_H) of 56 Å for the IgG monomer. The aggregation kinetics of the protein was followed by monitoring the time evolution of τ_D under conditions in which its cysteine residues were either free or blocked. In both cases, the progress curves confirmed that aggregation proceeded via the nucleation‐dependent polymerization pathway. However, for aggregation in the presence of free cysteines, the lag times were shorter, and the aggregate sizes bigger, than their respective counterparts for aggregation in the presence of blocked cysteines. This result clearly demonstrates that formation of intermolecular disulfide bonds represents a preferred pathway in the aggregation process of IgG. Fluorescence spectroscopy showed that aggregates formed in experiments where disulfide formation was prevented denatured at lower concentration of guanidine hydrochloride than those obtained in experiments where the disulfides were free to form, indicating that intermolecular disulfide bridging is a valid pathway for IgG aggregation. Proteins 2015; 83:169–177. © 2014 Wiley Periodicals, Inc.     

Early disulfide bond formation prevents heterotypic aggregation of membrane proteins in a cell-free translation system

We previously demonstrated that a heterotypic complex of the two rat asialoglycoprotein receptor subunits was assembled during cell-free translation (Sawyer, J. T., and D. Doyle. 1990. Proc. Natl. Acad. Sci. USA. 87:4854-4858). We have characterized this system further by analyzing polypeptide interactions under both reducing and oxidizing translation conditions. This report shows that the complex represents a heterogeneous interaction between reduced membrane proteins rather than a specific oligomeric structure. In the reduced state membrane proteins interact in this system to form aggregates of diverse size and composition. The aggregated nascent polypeptides interact with the immunoglobulin heavy chain binding protein but this protein is not an integral component of the aggregate. Aggregation occurs via the exoplasmic domain, rather than the transmembrane domain, and the folding of this domain by the formation of intramolecular disulfides, prevents the interaction from occurring. Additionally, the folded molecules containing intramolecular disulfides lack high affinity binding activity and thus appear to resemble the earliest folding intermediates seen in vivo (Olson, J. T., and M. D. Lane. 198. FASEB (Fed. Am. Soc. Exp. Biol.) J. 3:1618-1624). These results lead us to suggest that the formation of intramolecular disulfides during early biogenesis serves to prevent nonspecific associations between nascent polypeptides.     

Manipulating disulfide bond formation and protein folding in the endoplasmic reticulum.

Addition of the reducing agent dithiothreitol (DTT) to the medium of living cells prevented disulfide bond formation in newly synthesized influenza hemagglutinin (HA0) and induced the reduction of already oxidized HA0 inside the ER. The reduced HA0 did not trimerize or leave the ER. When DTT was washed out, HA0 was rapidly oxidized, correctly folded, trimerized and transported to the Golgi complex. We concluded that protein folding and the redox conditions in the ER can be readily manipulated by addition of DTT without affecting most other cellular functions, that the reduced influenza HA0 remains largely unfolded, and that folding events that normally take place on the nascent HA0 chains can be delayed and induced post-translationally without loss in efficiency.     

Preparation of protein conjugates via intermolecular disulfide bond formation

Conjugates of two unlike proteins can be prepared via the intermolecular disulfide interchange reaction, namely, protein A containing thiol groups reacts with protein B containing 4-dithiopyridyl groups to yield a conjugate with the release of 4-thiopyridone. Thiol groups can be introduced into proteins upon amidination with methyl 3-mercaptopropionimidate ester or 2-iminothiolane, and 4-dithiopyridyl groups can be introduced into proteins with these same reagents in the presence of 4,4'-dithiodipyridine. 2-Iminothiolane is stable on storage in contrast to the known lability of imidate esters; therefore 2-iminothiolane is a more convenient reagent for the modification of protein than are the imidate esters. All the reactions can be carried out easily under mild conditions in good yields. Conjugates of bovine plasma albumin with itself, ribonuclease, or a copolymer of D-glutamic acid and D-lysine and of sheep antibody and horseradish peroxidase were prepared with modified proteins containing an average of 1 to 5 thiol or dithiopyridyl groups per mol. These conjugates formed mainly dimers, trimers, and tetramers. The peroxidase labeled antibody retained more than 80% of its enzymatic and antigenic binding activities.     

Disulphide bridge formation of proinsulin fusion proteins during secretion in Streptomyces

To study disulphide bridge formation by Streptomyces lividans TK 24 in secreted single chain precursors of insulin a fusion protein (PTF 1) was investigated consisting of monkey proinsulin and the aminoterminal sequence Asp1 to Gly43 of the alpha-amylase inhibitor tendamistat from Streptomyces tendae. The purified soluble protein PTF 1 has a molecular mass of 14.4 kDa. The primary structure was elucidated after digestion with lysyl endopeptidase and fragment analysis. In this system, disulphide bond formation occurs in a way that the first cysteine in proinsulin is linked to the next following cysteine in the amino-acid chain resulting in a non-natural folding of the insulin part of the fusion protein. Re-folding of PTF 1 by reduction and re-oxidation followed by proteolytic digestions led to insulins which are identical to authentic material. The ease of correct disulphide bond formation in solution and incorrect processing during secretion suggests involvement of yet unknown factors leading to an unfavourable folding of proinsulin.     

Identification of a protein required for disulfide bond formation in vivo

We describe a mutation (dsbA) that renders Escherichia coli severely defective in disulfide bond formation. In dsbA mutant cells, pulse-labeled beta-lactamase, alkaline phosphatase, and OmpA are secreted but largely lack disulfide bonds. These disulfideless proteins may represent in vivo folding intermediates, since they are protease sensitive and chase slowly into stable oxidized forms. The dsbA gene codes for a 21,000 Mr periplasmic protein containing the sequence cys-pro-his-cys, which resembles the active sites of certain disulfide oxidoreductases. The purified DsbA protein is capable of reducing the disulfide bonds of insulin, an activity that it shares with these disulfide oxidoreductases. Our results suggest that disulfide bond formation is facilitated by DsbA in vivo.     

Dissimilar aggregation processes govern precipitation and gelation of human IgM cryoglobulins

Cryoglobulinemia is associated with a range of diseases including rheumatoid arthritis, B-cell malignancies, and chronic viral infections. This "cold-sensitivity" condition is caused by cryoglobulins that precipitate, gel, or occasionally crystallize in the cold. Clinical manifestations vary widely in severity, depending on many factors, including the type of cryoglobulin (monoclonal or mixed immunoglobulins) and the physical nature of the aggregates (precipitate, gel, or crystal). Dynamic light scattering (DLS) was used to examine the cold-induced precipitation or gelation of two human cryoglobulins, namely, Pot IgM and Yvo IgM. The DLS assay was highly reproducible, sensitive, and had low intra-assay variations for both IgM cryoglobulins. Distinct processes were revealed to contribute to precipitation and gelation of cryoglobulins. The precipitation of Pot IgM displayed a rapid transition from solution to solid phases, with a wide distribution of aggregate sizes. In contrast, the gelation of Yvo IgM progressed gradually across a broad temperature range to produce a relatively uniform gel matrix. Initial cryoglobulin concentrations determined the kinetics and critical temperatures for both precipitation and gelation. Moreover, the Yvo IgM was observed to have a distinct relationship between concentrations and mean hydrodynamic diameters or particle sizes. Concentration-dependent effects on particle sizes were present, but not as pronounced for the Pot IgM. Precipitation and gelation of cryoglobulins were also found to be differentially responsive to changes in the aqueous environment. Our results indicate that DLS is a rapid, reliable, and sensitive method for characterizing the nature of disease-associated cryoglobulins.     

Thermally induced protein gelation: gelation and rheological characterization of highly concentrated ovalbumin and soybean protein gels

In order to optimize the use of proteins as functional ingredients in foods, one needs more insight into the effects of environmental conditions (pH, ionic strength, and temperature) on the functional properties of protein. This paper summarizes the results of an extensive study on heat-induced gelation of ovalbumin (egg-white protein) and soybean protein in the concentration range from 10 to 35 g/100 g. It was the aim of the study to relate the rheological properties of thermally induced protein gels to the microstructure of the gel and the physicochemical properties of the constituent protein. The gelling behavior of the protein was quantified with rheological techniques, and the physical properties of the gels were determined, at small and large deformations. From the swelling/dissolving behavior of the gels in various media, the nature of the crosslinks was determined qualitatively. The microstructure of the gels was determined with electron microscopy. Nmr-spectroscopy was applied in order to elucidate changes in conformation during heating. It was found that the formation of a continuous covalently crosslinked network is not a prerequisite for thermally-induced protein gelation. The properties of a gel strongly depend on the pH at which the gel is formed. When heat-set at high pH(pH～10), a homogeneous, strong, and almost transparent gel is formed, consisting of flexible crosslinked protein gels. Heat-setting at low pH (pH 5) leads to the formation of a heterogeneous and weak gel, which easily exudes water. This gel consists of crosslinked aggregated protein. The ionic strength of the solvent in which the protein is dissolved and heat-set has a much lower effect on gel properties.     

Direct analysis of backbone-backbone hydrogen bond formation in protein folding transition states

Here we investigate the role of backbone-backbone hydrogen bonding interactions in stabilizing the protein folding transition states of two model protein systems, the B1 domain of protein L (ProtL) and the P22 Arc repressor. A backbone modified analogue of ProtL containing an amide-to-ester bond substitution between residues 105 and 106 was prepared by total chemical synthesis, and the thermodynamic and kinetic parameters associated with its folding reaction were evaluated. Ultimately, these parameters were used in a Phi-value analysis to determine if the native backbone-backbone hydrogen bonding interaction perturbed in this analogue (i.e. a hydrogen bond in the first beta-turn of ProtL's beta-beta-alpha-beta-beta fold) was formed in the transition state of ProtL's folding reaction. Also determined were the kinetic parameters associated with the folding reactions of two Arc repressor analogues, each containing an amide-to-ester bond substitution in the backbone of their polypeptide chains. These parameters were used together with previously established thermodynamic parameters for the folding of these analogues in Phi-value analyses to determine if the native backbone-backbone hydrogen bonding interactions perturbed in these analogues (i.e. a hydrogen bond at the end of the intersubunit beta-sheet interface and hydrogen bonds at the beginning of the second alpha-helix in Arc repressor's beta-alpha-alpha structure) were formed in the transition state of Arc repressor's folding reaction. Our results reveal that backbone-backbone hydrogen bonding interactions are formed in the beta-turn and alpha-helical transition state structures of ProtL and Arc repressor, respectively; and they were not formed in the intersubunit beta-sheet interface of Arc repressor, a region of Arc repressor's polypeptide chain previously shown to have other non-native-like conformations in Arc's protein folding transition state.     

Periplasmic transit and disulfide bond formation of the autotransported Shigella protein IcsA

The Shigella outer membrane protein IcsA belongs to the family of type V secreted (autotransported) virulence factors. Members of this family mediate their own translocation across the bacterial outer membrane: the carboxy-terminal beta domain forms a beta barrel channel in the outer membrane through which the amino-terminal alpha domain passes. IcsA, which is localized at one pole of the bacterium, mediates actin assembly by Shigella, which is essential for bacterial intracellular movement and intercellular dissemination. Here, we characterize the transit of IcsA across the periplasm during its secretion. We show that an insertion in the dsbB gene, whose gene product mediates disulfide bond formation of many periplasmic intermediates, does not affect the surface expression or unipolar targeting of IcsA. However, IcsA forms one disulfide bond in the periplasm in a DsbA/DsbB-dependent fashion. Furthermore, cellular fractionation studies reveal that IcsA has a transient soluble periplasmic intermediate. Our data also suggest that IcsA is folded in a proteinase K-resistant state in the periplasm. From these data, we propose a novel model for the secretion of IcsA that may be applicable to other autotransported proteins.