Oligomerization Equilibrium Research Articles

The Allosteric Regulation of Β-Ureidopropionase Depends on Fine-Tuned Stability of Active-Site Loops and Subunit Interfaces.

The activity of β-ureidopropionase, which catalyses the last step in the degradation of uracil, thymine, and analogous antimetabolites, is cooperatively regulated by the substrate and product of the reaction. This involves shifts in the equilibrium of the oligomeric states of the enzyme, but how these are achieved and result in changes in enzyme catalytic competence has yet to be determined. Here, the regulation of human β-ureidopropionase was further explored via site-directed mutagenesis, inhibition studies, and cryo-electron microscopy. The active-site residue E207, as well as H173 and H307 located at the dimer-dimer interface, are shown to play crucial roles in enzyme activation. Dimer association to larger assemblies requires closure of active-site loops, which positions the catalytically crucial E207 stably in the active site. H173 and H307 likely respond to ligand-induced changes in their environment with changes in their protonation states, which fine-tunes the active-site loop stability and the strength of dimer-dimer interfaces and explains the previously observed pH influence on the oligomer equilibrium. The correlation between substrate analogue structure and effect on enzyme assembly suggests that the ability to favourably interact with F205 may distinguish activators from inhibitors. The cryo-EM structure of human β-ureidopropionase assembly obtained at low pH provides first insights into the architecture of its activated state. and validates our current model of the allosteric regulation mechanism. Closed entrance loop conformations and dimer-dimer interfaces are highly conserved between human and fruit fly enzymes.

A not-glycosylated isoform of γ-conglutin, a hexameric glycoprotein of Lupinus albus seed, participates in the oligomerization equilibrium

γ-conglutin (γ-C) is a hexameric glycoprotein accumulated in lupin seeds and has long been considered as a storage protein. Recently, it has been investigated for its possible postprandial glycaemic regulating action in human nutrition and for its physiological role in plant defence. The quaternary structure of γ-C results from the assembly of six monomers in reversible pH-dependent association/dissociation equilibrium. Our working hypothesis was that the γ-C hexamer is made up of glycosylated subunits in association with not-glycosylated isoforms, that seem to have ‘escaped’ the correct glycosylation process in the Golgi. Here we describe the isolation of not-glycosylated γ-C monomers in native condition by two in tandem lectin-based affinity chromatography and the characterization of their oligomerization capacity. We report, for the first time, the observation that a plant multimeric protein may be formed by identical polypeptide chains that have undergone different post-translational modifications. All obtained considered, the results strongly suggest that the not-glycosylated isoform can also take part in the oligomerization equilibrium of the protein.

Application of particle swarm optimization to understand the mechanism of action of allosteric inhibitors of the enzyme HSD17β13

Understanding a drug candidate's mechanism of action is crucial for its further development. However, kinetic schemes are often complex and multi-parametric, especially for proteins in oligomerization equilibria. Here, we demonstrate the use of particle swarm optimization (PSO) as a method to select between different sets of parameters that are too far apart in the parameter space to be found by conventional approaches. PSO is based upon the swarming of birds: each bird in the flock assesses multiple landing spots while atthe same time sharing that information with its neighbors. We applied this approach to the kinetics ofHSD17β13 enzyme inhibitors, which displayed unusually large thermal shifts. Thermal shift data for HSD17β13 indicated that the inhibitor shifted the oligomerization equilibrium toward the dimeric state. Validation of the PSO approach was provided by experimental mass photometry data. These results encourage further exploration of multi-parameter optimization algorithms as tools in drug discovery.

Modulatory role of copper on hIAPP aggregation and toxicity in presence of insulin

Aggregation of the human islets amyloid polypeptide, or hIAPP, is linked to β-cell death in type II diabetes mellitus (T2DM). Different pancreatic β-cell environmental variables such as pH, insulin and metal ions play a key role in controlling the hIAPP aggregation. Since insulin and hIAPP are co-secreted, it is known from numerous studies that insulin suppresses hIAPP fibrillation by preventing the initial dimerization process. On the other hand, zinc and copper each have an inhibitory impact on hIAPP fibrillation, but copper promotes the production of toxic oligomers. Interestingly, the insulin oligomeric equilibrium is controlled by the concentration of zinc ions when the effect of insulin and zinc has been tested together. Lower zinc concentrations cause the equilibrium to shift towards the monomer and dimer states of insulin, which bind to monomeric hIAPP and stop it from developing into a fibril. On the other hand, the combined effects of copper and insulin have not yet been studied. In this study, we have demonstrated how the presence of copper affects hIAPP aggregation and the toxicity of the resultant conformers with or without insulin. For this purpose, we have used a set of biophysical techniques, including NMR, fluorescence, CD etc., in combination with AFM and cell cytotoxicity assay. In the presence and/or absence of insulin, copper induces hIAPP to form structurally distinct stable toxic oligomers, deterring the fibrillation process. More specifically, the oligomers generated in the presence of insulin have slightly higher toxicity than those formed in the absence of insulin. This research will increase our understanding of the combined modulatory effect of two β-cell environmental factors on hIAPP aggregation.

Probing Oxidant Effects on Superoxide Dismutase 1 Oligomeric States in Live Cells Using Single-Molecule Fluorescence Anisotropy.

The protein Cu/Zn superoxide dismutase (SOD1) is known to function as a dimer, but its concentration in cells (∼50 μM) and the dimerization constant (K d of 500 μM) results suggest that it exists in a monomer-dimer equilibrium. It is unclear how the oligomeric state of SOD1 changes when cells are initially exposed to high levels of extracellular oxidative stress. To address this problem, we introduced the single-molecule fluorescence anisotropy (smFA) assay to explore SOD1 oligomeric states in live COS7 cells. smFA specifically probes the fluorescence polarization changes caused by molecular rotations where the fast-rotating molecules (either due to smaller hydrodynamic volume or less viscous environments) deteriorate the emission polarization and thus lower the anisotropy. After validating that smFA is effective in distinguishing monomeric and dimeric fluorescence proteins, we overexpressed SOD1 in live COS7 cells and investigated how its oligomeric state changes under basal, 2 h, and 24 h 100 μM H2O2 treatments. We found that treating cells with H2O2 promotes SOD1 dimerization and decreases cellular viscosity in 2 h. Interestingly, prolonged H2O2 treatments show similar results as the basal conditions, indicating that cells return to a steady state similar to the basal state after 24 h, despite the presence of H2O2. Our results demonstrate that SOD1 changes its oligomeric state equilibrium in response to extracellular oxidative stresses. smFA will open new opportunities to explore the relationship between the SOD1 oligomer state and its H2O2-based signaling and transcription regulation roles.

Biophysical evaluation of the oligomerization and conformational properties of the N-terminal domain of TDP-43

TDP-43 is an RNA-binding protein that presents four domains comprising an N-terminal region, two RNA recognition motifs and a C-terminal region. The N-terminal domain (NTD) has a relevant role in the oligomerization and splicing activity of TDP-43. In this work, we have expressed, purified and biophysically characterized the region that includes residues 1 to 102 that contains the nuclear localization signal (residues 80–102, NLS). Furthermore, we have evaluated the oligomerization equilibrium for this protein fragment. Also, we have determined changes in the tertiary structure and its stability in a broad range of pH values by means of different spectroscopic methods. Additionally, we compared this fragment with the one that lacks the NLS employing experimental and computational methods. Finally, we evaluated the motion of dimeric forms to get insights into the conformational flexibility of this TDP-43 module in an oligomeric state. Our results suggest that this domain has a conformational plasticity in the vicinity of the single tryptophan of this domain (Trp68), which is enhanced by the presence of the nuclear localization signal. All these results help to understand the molecular features of the NTD of TDP-43.

Thermodynamic and kinetic analysis on oligomeric protein dissociation using high-pressure native PAGE velocity method

High pressure is known to dissociate several oligomeric proteins, and regarded as an important tool to shift the oligomerization equilibrium. Native polyacrylamide gel electrophoresis (native PAGE) at high pressure can characterize the dissociates and clearly discriminate the aggregates. However, a band smearing of migration profiles often hinders more detailed analyses (Miwa et al., High Pressure Res. (2019) 39, 218–224). In this paper, we focused on the band smearing dependent on the migration velocity so as to extract both thermodynamic and kinetic parameters. We systematically perturbed the migration velocity by changing the gel concentration and carried out numerical analysis for a series of the migration profiles based on a simple dissociation reaction scheme with limited thermodynamic and kinetic parameters. Then, complete volumetric properties on oligomerization process can be available. We term the present analysis method as a high-pressure native PAGE velocity method. We also report the application of this method to revisit the pressure dissociation of tetrameric lactate dehydrogenase (LDH) from pig heart.

A finely tuned interplay between calcium binding, ionic strength and pH modulates conformational and oligomerization equilibria in the Respiratory Syncytial Virus Matrix (M) protein

As in most enveloped RNA viruses, the Respiratory Syncytial Virus Matrix (RSV-M) protein plays key roles in viral assembly and uncoating. It also plays non-structural roles related to transcription modulation through nucleo-cytoplasmic shuttling and nucleic acid binding ability. We dissected the structural and conformational changes underlying the switch between multiple functionalities, identifying Ca2+ binding as a key factor. To this end, we tackled the analysis of M's conformational stability and equilibria. While in silico calculations predict two potential calcium binding sites per protomer, purified RSV-M dimer contains only one strongly bound calcium ion per protomer. Incubation of RSV-M in the presence of excess Ca2+ leads to an increase in the thermal stability, confirming additional Ca2+ binding sites. Moreover, mild denaturant concentrations trigger the formation of higher order oligomers which are otherwise prevented under Ca2+ saturation conditions, in line with the stabilizing effect of the additional low affinity binding site. On the other hand, Ca2+ removal by chelation at pH 7.0 causes a substantial decrease in the thermal stability leading to the formation of amorphous, spherical-like aggregates, as assessed by TEM. Even though the Ca2+ content modulates RSV-M oligomerization propensity, it does affect its weak RNA binding ability. RSV-M undergoes a substantial conformational change at pHs 4.0 to 5.0 that results in the exposure of hydrophobic surfaces, an increase beta sheet content but burial of tryptophan residues. While low ionic strength promotes dimer dissociation at pH 4.0, physiological concentrations of NaCl lead to the formation of soluble oligomers smaller than 400 kDa at pH 4.0 or insoluble aggregates with tubular morphology at pH 5.0, supporting a fine tuning by pH. Furthermore, the dissociation constants estimated for the low- and high affinity calcium binding sites are 13 μM and 58 nM, respectively, suggesting an intracellular calcium sensing mechanism of RSV-M upon infection.We uncover a finely tuned interplay between calcium binding, ionic strength, and pH changes compatible with the different cellular compartments where M plays key roles, revealing diverse conformational equilibria, oligomerization, and high order structures, required to stabilize the virion particle by a layer of molecules positioned between the membrane and the nucleocapsid.

Dilute phase oligomerization can oppose phase separation and modulate material properties of a ribonucleoprotein condensate

SignificanceA large subclass of biomolecular condensates are linked to RNA regulation and are known as ribonucleoprotein (RNP) bodies. While extensive work has identified driving forces for biomolecular condensate formation, relatively little is known about forces that oppose assembly. Here, using a fungal RNP protein, Whi3, we show that a portion of its intrinsically disordered, glutamine-rich region modulates phase separation by forming transient alpha helical structures that promote the assembly of dilute phase oligomers. These oligomers detour Whi3 proteins from condensates, thereby impacting the driving forces for phase separation, the protein-to-RNA ratio in condensates, and the material properties of condensates. Our findings show how nanoscale conformational and oligomerization equilibria can influence mesoscale phase equilibria.

Impact of Fluorinated Ionic Liquids on Human Phenylalanine Hydroxylase-A Potential Drug Delivery System.

Phenylketonuria (PKU) is an autosomal recessive disease caused by deficient activity of human phenylalanine hydroxylase (hPAH), which can lead to neurologic impairments in untreated patients. Although some therapies are already available for PKU, these are not without drawbacks. Enzyme-replacement therapy through the delivery of functional hPAH could be a promising strategy. In this work, biophysical methods were used to evaluate the potential of [N1112(OH)][C4F9SO3], a biocompatible fluorinated ionic liquid (FIL), as a delivery system of hPAH. The results herein presented show that [N1112(OH)][C4F9SO3] spontaneously forms micelles in a solution that can encapsulate hPAH. This FIL has no significant effect on the secondary structure of hPAH and is able to increase its enzymatic activity, despite the negative impact on protein thermostability. The influence of [N1112(OH)][C4F9SO3] on the complex oligomerization equilibrium of hPAH was also assessed.

Blocking PSD95-PDZ3's amyloidogenesis through point mutations that inhibit high-temperature reversible oligomerization (RO).

The third PDZ domain of the postsynaptic density protein 95 (PSD95-PDZ3; 11 kDa, 103 residues) has a propensity to form amyloid fibrils at high temperatures. At neutral pH, PDZ3 is natively folded, but it exhibits a peculiar three-state thermal unfolding with a reversible oligomerization (RO) equilibrium at high temperatures, which is uncharacteristic in the unfolding of a small globular protein as PDZ3 is. Here, we examined the RO's role in PDZ3's amyloidogenesis at high-temperature using two variants (F340A and L342A) that suppress the high-temperature RO and five single-alanine-mutated variants, where we mutated surface-exposed hydrophobic residues to alanine. Circular Dichroism (CD), Analytical Ultracentrifuge (AUC), and other spectroscopic measurements confirmed the retention of the native structure at ambient temperature. Differential Scanning Calorimetry (DSC) was used to assess the presence or absence of the high-temperature RO, and the amyloidogenicity of the variants was measured by Thioflavin T (ThT) fluorescence and Transmission Electron Microscopy (TEM). By comparing the fraction of RO and the ThT signal, we found that mutations that suppressed the high-temperature RO strongly inhibited amyloidogenesis. On the other hand, all variants forming RO also formed amyloids under the same conditions as the wild-type PDZ3.

An Eight Amino Acid Segment Controls Oligomerization and Preferred Conformation of the two Non-visual Arrestins

G protein coupled receptors signal through G proteins or arrestins. A long-standing mystery in the field is why vertebrates have two non-visual arrestins, arrestin-2 and arrestin-3. These isoforms are ~75% identical and 85% similar; each binds numerous receptors, and appear to have many redundant functions, as demonstrated by studies of knockout mice. We previously showed that arrestin-3 can be activated by inositol-hexakisphosphate (IP6). IP6 interacts with the receptor-binding surface of arrestin-3, induces arrestin-3 oligomerization, and this oligomer stabilizes the active conformation of arrestin-3. Here, we compared the impact of IP6 on oligomerization and conformational equilibrium of the highly homologous arrestin-2 and arrestin-3 and found that these two isoforms are regulated differently. In the presence of IP6, arrestin-2 forms “infinite” chains, where each promoter remains in the basal conformation. In contrast, full length and truncated arrestin-3 form trimers and higher-order oligomers in the presence of IP6; we showed previously that trimeric state induces arrestin-3 activation (Chen et al., 2017). Thus, in response to IP6, the two non-visual arrestins oligomerize in different ways in distinct conformations. We identified an insertion of eight residues that is conserved across arrestin-2 homologs, but absent in arrestin-3 that likely accounts for the differences in the IP6 effect. Because IP6 is ubiquitously present in cells, this suggests physiological consequences, including differences in arrestin-2/3 trafficking and JNK3 activation. The functional differences between two non-visual arrestins are in part determined by distinct modes of their oligomerization. The mode of oligomerization might regulate the function of other signaling proteins.

Proteinaceous Transformers: Structural and Functional Variability of Human sHsps.

The proteostasis network allows organisms to support and regulate the life cycle of proteins. Especially regarding stress, molecular chaperones represent the main players within this network. Small heat shock proteins (sHsps) are a diverse family of ATP-independent molecular chaperones acting as the first line of defense in many stress situations. Thereby, the promiscuous interaction of sHsps with substrate proteins results in complexes from which the substrates can be refolded by ATP-dependent chaperones. Particularly in vertebrates, sHsps are linked to a broad variety of diseases and are needed to maintain the refractive index of the eye lens. A striking key characteristic of sHsps is their existence in ensembles of oligomers with varying numbers of subunits. The respective dynamics of these molecules allow the exchange of subunits and the formation of hetero-oligomers. Additionally, these dynamics are closely linked to the chaperone activity of sHsps. In current models a shift in the equilibrium of the sHsp ensemble allows regulation of the chaperone activity, whereby smaller oligomers are commonly the more active species. Different triggers reversibly change the oligomer equilibrium and regulate the activity of sHsps. However, a finite availability of high-resolution structures of sHsps still limits a detailed mechanistic understanding of their dynamics and the correlating recognition of substrate proteins. Here we summarize recent advances in understanding the structural and functional relationships of human sHsps with a focus on the eye-lens αA- and αB-crystallins.

The R882H DNMT3A hot spot mutation stabilizes the formation of large DNMT3A oligomers with low DNA methyltransferase activity

DNMT3A (DNA methyltransferase 3A) is a de novo DNA methyltransferase responsible for establishing CpG methylation patterns within the genome. DNMT3A activity is essential for normal development, and its dysfunction has been linked to developmental disorders and cancer. DNMT3A is frequently mutated in myeloid malignancies with the majority of mutations occurring at Arg-882, where R882H mutations are most frequent. The R882H mutation causes a reduction in DNA methyltransferase activity and hypomethylation at differentially-methylated regions within the genome, ultimately preventing hematopoietic stem cell differentiation and leading to leukemogenesis. Although the means by which the R882H DNMT3A mutation reduces enzymatic activity has been the subject of several studies, the precise mechanism by which this occurs has been elusive. Herein, we demonstrate that in the context of the full-length DNMT3A protein, the R882H mutation stabilizes the formation of large oligomeric DNMT3A species to reduce the overall DNA methyltransferase activity of the mutant protein as well as the WT-R882H complex in a dominant-negative manner. This shift in the DNMT3A oligomeric equilibrium and the resulting reduced enzymatic activity can be partially rescued in the presence of oligomer-disrupting DNMT3L, as well as DNMT3A point mutations along the oligomer-forming interface of the catalytic domain. In addition to modulating the oligomeric state of DNMT3A, the R882H mutation also leads to a DNA-binding defect, which may further reduce enzymatic activity. These findings provide a mechanistic explanation for the observed loss of DNMT3A activity associated with the R882H hot spot mutation in cancer.

His6, His13, and His14 residues in A\u03b2 1\u201340 peptide significantly and specifically affect oligomeric equilibria

Oligomers of Aβ peptide are implicated as the most probable causative agent in Alzheimer’s disease. However, their structural properties remain elusive due to the dynamic and heterogeneous character of oligomeric species coexisting in solution. Nevertheless, new approaches, mainly based on mass spectrometry, provide unique access to these different structural forms. Using these methods, we previously showed that the N-terminal, non-amyloidogenic region of Aβ is involved in the network of interactions specifically stabilizing oligomers. In the present study, we identified three histidine residues as active participants in this network. Detailed knowledge of the structural features that are potentially important for oligomer-mediated neurotoxicity is a prerequisite for the rational design of oligomerization modifiers.

Substrate-induced structural alterations of Mycobacterial mycothione reductase and critical residues involved.

Redox homeostasis is a prerequisite for survival of the pathogen Mycobacterium tuberculosis (Mtb) which employs the low molecular weight thiol mycothiol (MSH). The Mycobacterial NADPH-dependent mycothione reductase (MtMtr), composed of an NADPH-, FAD-, and a dimerization-domain connected by linkers, regulates the balance of oxidized-reduced MSH. Here, we demonstrate by small-angle X-ray scattering, that NADPH-binding alters the oligomeric state equilibrium of the protein with no significant overall structural change after MSH-binding. Mutation of critical residues in the linker regions of MtMtr eliminate partially or totally the NADPH-induced oligomerization effect with simultaneous effect on enzyme activity. The data provide insight into the MtMtr linker regions involved in the novel oligomerization equilibrium of the Mycobacterial enzyme.

Chaperonin of Group I: Oligomeric Spectrum and Biochemical and Biological Implications.

Chaperonins play various physiological roles and can also be pathogenic. Elucidation of their structure, e.g., oligomeric status and post-translational modifications (PTM), is necessary to understand their functions and mechanisms of action in health and disease. Group I chaperonins form tetradecamers with two stacked heptameric rings. The tetradecamer is considered the typical functional complex for folding of client polypeptides. However, other forms such as the monomer and oligomers with smaller number of subunits than the classical tetradecamer, also occur in cells. The properties and functions of the monomer and oligomers, and their roles in chaperonin-associated diseases are still incompletely understood. Chaperonin I in eukaryotes occurs in various locations, not just the mitochondrion, which is its canonical place of residence and function. Eukaryotic Chaperonin I, namely Hsp60 (designated HSP60 or HSPD1 in humans) has, indeed, been found in the cytosol; the plasma-cell membrane; on the outer surface of cells; in the intercellular space; in biological liquids such as lymph, blood, and cerebrospinal fluid; and in secretions, for instance saliva and urine. Hsp60 has also been found in cell-derived vesicles such as exosomes. The functions of Hsp60 in all these non-canonical locales are still poorly characterized and one of the questions not yet answered is in what form, i.e., monomer or oligomer, is the chaperonin present in these non-canonical locations. In view of the steady increase in interest on chaperonopathies over the last several years, we have studied human HSP60 to determine its role in various diseases, its locations in cells and tissues and migrations in the body, and its post-translational modifications that might have an impact on its location and function. We also carried out experiments to characterize the oligomeric status of extramitochondrial of HSP60 in solution. Here, we provide an overview of our results, focusing on the oligomeric equilibrium and stability of the various forms of HSP60 in comparison with GroEL. We also discuss post-translational modifications associated with anti-cancer drugs to indicate the potential of Hsp60 in Medicine, as a biomarker and etiopathogenic factor.

Biochemical Basis for Distinct Roles of the Heterochromatin Proteins Swi6 and Chp2

Heterochromatin protein 1 (HP1) family proteins are conserved chromatin binding proteins involved in gene silencing, chromosome packaging, and chromosome segregation. These proteins recognize histone H3 lysine 9 methylated tails via their chromodomain and recruit additional ligand proteins with diverse activities through their dimerization domain, the chromoshadow domain. Species that have HP1 proteins possess multiple paralogs that perform non-overlapping roles in vivo. How different HP1 proteins, which are highly conserved, perform different functions is not well understood. Here, we use the two Schizosaccharomyces pombe HP1 paralogs, Swi6 and Chp2, as model systems to compare and contrast their biophysical properties. We find that Swi6 and Chp2 have similar dimerization and oligomerization equilibria, and that Swi6 binds slightly (~3-fold) more strongly to nucleosomes than Chp2. Furthermore, while Swi6 binding to the H3K9me3 mark is regulated by a previously described auto-inhibition mechanism, the binding of Chp2 to the H3K9me3 mark is not analogously regulated. In the context of chromoshadow domain interactions, we show using a newly identified peptide sequence from the Clr3 histone deacetylase and a previously identified sequence from the protein Shugoshin that the Swi6 chromoshadow domain binds both ligands more strongly than the Chp2. Overall, our findings uncover quantitative differences in how Swi6 and Chp2 interact with nucleosomal and non-nucleosomal ligands and qualitative differences in how their assembly on nucleosomes is regulated. These findings provide a biochemical framework to explain the varied functions of Chp2 and Swi6 in vivo.

An Electrospray Ionization Mass Spectrometry Study on the "In Vacuo" Hetero-Oligomers Formed by the Antimicrobial Peptides, Surfactin and Gramicidin S.

It was previously observed that the lipopeptide surfactants in surfactin (Srf) have an antagonistic action towards the highly potent antimicrobial cyclodecapeptide, gramicidin S (GS). This study reports on some of the molecular aspects of the antagonism as investigated through complementary electrospray ionization mass spectrometry techniques. We were able to detect stable 1:1 and 2:1 hetero-oligomers in a mixture of surfactin and gramicidin S. The noncovalent interaction between GS and Srf, with the proposed equilibrium: GS~Srf↔GS+Srf correlated to apparent K d values of 6-9 μM in gas-phase and 1 μM in aqueous solution. The apparent K d values decreased with a longer incubation time and indicated a slow oligomerization equilibrium. Furthermore, the low μM K dapp values of GS~Srf↔GS+Srf fell within the biological concentration range and related to the 2- to 3-fold increase in [GS] needed for bacterial growth inhibition in the presence of Srf. Competition studies indicated that neither Na+ nor Ca2+ had a major effect on the stability of preformed heterodimers and that GS in fact out-competed Ca2+ and Na+ from Srf. Traveling wave ion mobility mass spectrometry revealed near symmetrical peaks of the heterodimers correlating to a compact dimer conformation thatdepend on specific interactions. Collision-induced dissociation studies indicated that the peptide interaction is most probably between one Orn residue in GS and the Asp residue, but not the Glu residue in Srf. We propose that flanking hydrophobic residues in both peptides stabilize the antagonistic and inactive peptide hetero-oligomers and shield the specific polar interactions in an aqueous environment. Graphical Abstract ᅟ.

Effect of N-terminal region of nuclear Drosophila melanogaster small heat shock protein DmHsp27 on function and quaternary structure.

The importance of the N-terminal region (NTR) in the oligomerization and chaperone-like activity of the Drosophila melanogaster small nuclear heat shock protein DmHsp27 was investigated by mutagenesis using size exclusion chromatography and native gel electrophoresis. Mutation of two sites of phosphorylation in the N-terminal region, S58 and S75, did not affect the oligomerization equilibrium or the intracellular localization of DmHsp27 when transfected into mammalian cells. Deletion or mutation of specific residues within the NTR region delineated a motif (FGFG) important for the oligomeric structure and chaperone-like activity of this sHsp. While deletion of the full N-terminal region, resulted in total loss of chaperone-like activity, removal of the (FGFG) at position 29 to 32 or single mutation of F29A/Y, G30R and G32R enhanced oligomerization and chaperoning capacity under non-heat shock conditions in the insulin assay suggesting the importance of this site for chaperone activity. Unlike mammalian sHsps DmHsp27 heat activation leads to enhanced association of oligomers to form large structures of approximately 1100 kDa. A new mechanism of thermal activation for DmHsp27 is presented.