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PubMed2000

Effects of side-chain characteristics on stability and oligomerization state of a de novo-designed model coiled-coil: 20 amino acid substitutions in position "d".

We describe the de novo design and biophysical characterization of a model coiled-coil protein in which we have systematically substituted 20 different amino acid residues in the central "d" position. The model protein consists of two identical 38 residue polypeptide chains covalently linked at their N termini via a disulfide bridge. The hydrophobic core contained Val and Ile residues at positions "a" and Leu residues at positions "d". This core allowed for the formation of both two-stranded and three-stranded coiled-coils in benign buffer, depending on the substitution at position "d". The structure of each analog was analyzed by CD spectroscopy and their relative stability determined by chemical denaturation using GdnHCI (all analogs denatured from the two-stranded state). The oligomeric state(s) was determined by high-performance size-exclusion chromatography and sedimentation equilibrium analysis in benign medium. Our results showed a thermodynamic stability order (in order of decreasing stability) of: Leu, Met, Ile, Tyr, Phe, Val, Gln, Ala, Trp, Asn, His, Thr, Lys, Ser, Asp, Glu, Arg, Orn, and Gly. The Pro analog prevented coiled-coil formation. The overall stability range was 7.4 kcal/mol from the lowest to the highest analog, indicating the importance of the hydrophobic core and the dramatic effect a single substitution in the core can have upon the stability of the protein fold. In general, the side-chain contribution to the level of stability correlated with side-chain hydrophobicity. Molecular modelling studies, however, showed that packing effects could explain deviations from a direct correlation. In regards to oligomerization state, eight analogs demonstrated the ability to populate exclusively one oligomerization state in benign buffer (0.1 M KCl, 0.05 M K(2)PO(4)(pH 7)). Ile and Val (the beta-branched residues) induced the three-stranded oligomerization state, whereas Tyr, Lys, Arg, Orn, Glu and Asp induced the two-stranded state. Asn, Gln, Ser, Ala, Gly, Phe, Leu, Met and Trp analogs were indiscriminate and populated two-stranded and three-stranded states. Comparison of these results with similar substitutions in position "a" highlights the positional effects of individual residues in defining the stability and numbers of polypeptide chains occurring in a coiled-coil structure. Overall, these results in conjunction with other work now generate a relative thermodynamic stability scale for 19 naturally occurring amino acid residues in either an "a" or "d" position of a two-stranded coiled-coil. Thus, these results will aid in the de novo design of new coiled-coil structures, a better understanding of their structure/function relationships and the design of algorithms to predict the presence of coiled-coils within native protein sequences.

Algorithms↗
PubMed2000

Two different oligomeric states of the RuvB branch migration motor protein as revealed by electron microscopy.

In prokaryotes, the RuvA, B, and C proteins play major roles at the late stage of DNA homologous recombination, where RuvB complexed with RuvA acts as an ATP-dependent motor for branch migration. The oligomeric structures of negatively stained and frozen hydrated RuvB from Thermus thermophilus HB8 were investigated by electron microscopy. RuvB oligomers free of DNA formed a ring structure of about 14 nm in diameter. The averaged top view image clearly indicated a sevenfold symmetry, suggesting that it exists as a heptamer. The RuvB oligomers complexed with duplex DNA formed a smaller ring of about 13 nm in diameter. The averaged top view images represented a sixfold symmetry. This difference in oligomerization indicates that the oligomeric structure of RuvB may convert from a heptamer to a hexamer upon DNA binding. In addition, this finding provides the lesson that great care should be taken in investigating the subunit organizations of DNA binding proteins, because their oligomeric states are more sensitive to DNA interactions than expected.

Adenosine Triphosphatases↗
PubMed1997

Oligomerization of HIV-1 Rev mutants in the cytoplasm and during nuclear import.

Oligomerization of Rev molecules has been shown to be required for Rev function. In addition to a Western blot assay monitoring dimer formation, a new in vivo assay analyzing formation of Rev heteromers in the cytoplasm and during nuclear import is presented here. The oligomerization assay is based upon the ability of Rev mutants with an intact nuclear localization signal (NLS) to interact specifically with mutants with a defective NLS and translocate such mutants to the nuclear compartments. Several of the mutants previously reported to be oligomerization defective were found to mediate nuclear and nucleolar localization of the NLS mutant. The Rev mutant previously named M4 was the only mutant tested that did not translocate the mutant with a defective NLS to the nucleus. Furthermore, the predominantly cytoplasmic localization of the M4 mutant suggests that oligomerization is important for effective nuclear import of Rev.

Animals↗
PubMed2002

Characterization of the oligomerization domain of the phosphoprotein of human parainfluenza virus type 3.

The phosphoprotein (P) of human parainfluenza virus type 3 (HPIV 3) plays a central role in the viral genome RNA transcription and replication. It acts as an essential cofactor of the RNA polymerase (L) by forming a functional L-P complex, binds to the genomic N-RNA template to recruit the L-P complex for RNA synthesis, and interacts with the nucleocapsid protein (N) to form the encapsidation complex (N-P). We have earlier demonstrated that the P protein forms oligomers (B. P. De, M. A. Hoffman, S. Choudhary, C. C. Huntley, and A. K. Banerjee, 2000, J. Virol. 74, 5886-5895) and in this article we identified the putative oligomerization domain of the P protein and studied the role of this domain in transcription. By computer analyses, we have localized a high-score coiled-coil motif characteristic of oligomerization domain residing between the amino acid residues 423 and 457 of the P protein. Deletion of 12 amino acid residues within this coiled-coil motif (P Delta 439-450) completely abrogated oligomerization, whereas deletion in other regions outside the motif had no significant effect. The mutant P Delta 439-450 was both defective in mRNA synthesis in vitro and minigenome transcription in vivo. Interestingly, the mutant interacted with L to form L-P complex, albeit less efficiently, while its interaction with N protein to form N-P complex and with N-RNA template was similar to the wt P protein. Our results indicate that oligomerization provides a key function to the P protein in the transcription of HPIV 3 genome RNA.

Amino Acid Motifs↗
PubMed1990

Erythrocyte Ca2(+)-ATPase: activation by enzyme oligomerization versus by calmodulin.

The subject of our studies is the mechanism of activation of the erythrocyte Ca2(+)-ATPase. Using purified, detergent solubilized enzyme it was found that equivalent maximal Ca2(+)-ATPase activity is obtained either upon addition of calmodulin or upon increase of enzyme concentration. Three independent methods, including Ca2(+)-ATPase activity, polarization of the enzyme modified with an external fluorescent probe, and efficiency of fluorescence resonance energy transfer between enzyme molecules have established that the concentration dependent activation is due to enzyme oligomerization. The oligomers bind calmodulin with a lower stoichiometry (0.5 mol calmodulin/mol Ca2(+)-ATPase), higher Ca2+ affinity (KCa = pCa 7.4), and higher cooperativity for Ca2+ (nH = 2.6) than the monomeric form (stoichiometry = 1 mol calmodulin/mol Ca2(+)-ATPase, KCa = pCa 7.0, nH = 1.1). The Ca2+ dependence of calmodulin binding and activation of monomers indicates that calmodulin binds before the Ca2(+)-ATPase activity is exhibited, demonstrating that the activation of this enzyme form is totally dependent on calmodulin. In contrast, oligomers reveal very similar Ca2+ dependence for calmodulin binding and for Ca2(+)-ATPase activity as well as for Ca2+ binding (assessed by tryptophan fluorescence), and for the oligomerization process (assessed by fluorescence energy transfer). The calmodulin antagonist drug 48/80 inhibits the calmodulin dependent activity of the monomers (I50 = 1.4 micrograms/ml) but has no effect on the activity of oligomers, confirming that calmodulin plays no role in the activation of the oligomeric enzyme. Our studies indicate that the erythrocyte Ca2(+)-ATPase can be activated by its high affinity, Ca2+ dependent binding of calmodulin or by a Ca2+ dependent oligomerization process which may involve calmodulin binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1994

Characterization of different oligomeric species of the Yersinia enterocolitica outer membrane protein YadA.

The oligomeric structure of the plasmid-encoded outer membrane protein YadA of Yersinia enterocolitica was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and sucrose gradient sedimentation, respectively. The apparent molecular weight (M(r)) of the oligomeric 200-kDa YadA species detected by SDS-PAGE varied from 152,000 to 240,000 depending on the respective acrylamide concentration. The atypical electrophoretic behavior of the 200-kDa YadA species results form an exceptionally high relative free mobility as revealed by the Ferguson plot. In contrast, the apparent M(r) of 53,000 of the YadA monomer was independent of the acrylamide concentration. An additional oligomeric 116-kDa YadA species was detected by SDS-PAGE when membrane preparations of Y. enterocolitica were solubilized in SDS at 37 degrees C. The gel-purified 116-kDa YadA species was completely converted to the 200-kDa species by heating at 100 degrees C and to the monomeric form (M(r) 53,000) by heating in the presence of 10 M urea without reducing agents, respectively. This suggests that the 116-kDa YadA species represents the native oligomeric form of YadA, whereas the 200-kDa species is only generated from native YadA during denaturation in SDS. The significance of the 116-kDa YadA species is also supported by the rather slow sedimentation at about 6 S of detergent-solubilized YadA in sucrose gradients, which probably contains only two or three monomers.

Adhesins, Bacterial↗
PubMed2003

Display of a functional hetero-oligomeric catalytic antibody on the yeast cell surface.

A functional hetero-oligomeric protein was, for the first time, displayed on the yeast cell surface. A hetero-oligomeric Fab fragment of the catalytic antibody 6D9 can hydrolyze a non-bioactive chloramphenicol monoester derivative to produce chloramphenicol. The gene encoding the light chain of the Fab fragment of 6D9 was expressed with the tandemly-linked C-terminal half of alpha-agglutinin. At the same time, the gene encoding the Fd fragment of the heavy chain of the Fab fragment was expressed as a secretion protein. The combined Fab fragment displayed and associated on the yeast cell surface had an intermolecular disulfide linkage between the light and heavy chains. This protein fragment catalyzed the hydrolysis of a chloramphenicol monoester derivative and exhibited high stability in binding with a transition-state analog (TSA). The catalytic reaction was also inhibited by the TSA. The successful display of a functional hetero-oligomeric catalytic antibody provides a useful model for the display of hetero-oligomeric proteins and enzymes.

Antibodies, Catalytic↗
PubMed2000

Molecular design of novel metal-binding oligomeric human metallothioneins.

Genes for dimeric and tetrameric human metallothionein (hMT) were designed and successfully overexpressed in Escherichia coli to generate functional oligomeric hMTs. An hMT synthesized with prokaryotic codons, a linker encoding a gly-gly-gly tripeptide, and Met-deficient hMT-II was ligated to create a dimeric hMT, from which a tetrameric hMT was then constructed. The increased molecular size of the constructs resulted in improved stability and productivity in E. coli. The oligomeric proteins formed inclusion bodies which were dissolved with dithiothreitol, and the purified apometallothioneins were reconstituted with Cd or Zn ions in a reducing condition. The oligomeric hMT proteins incubated with Cd ions showed a typical Cd-thiolate absorbance peak at 245-255 nm. The dimeric and tetrameric hMT proteins exhibited both Cd and Zn binding activities that were respectively two and four times higher than those of the hMT-II monomer protein. These novel oligomeric hMTs may be useful in bioremediation for heavy metals.

Amino Acid Sequence↗
PubMed2002

Diffusion of beta-lactam antibiotics through oligomeric or monomeric porin channels of some gram-negative bacteria.

The penetration of anionic beta-lactam antibiotics through porins was evaluated as a mechanism of drug resistance. The major proteins with porin activity were purified from the outer membranes of six bacteria. Three of the six porins were oligomeric porins. The molecular weights of their monomers were 37 kDa from Photobacterium damsela, 42 kDa from Serratia liquefaciens, and 36 kDa from E. coli B. The other three porins were heat-modifiable monomeric porins with molecular weights of 43 kDa from Porphyromonas asaccharolytica and Acinetobacter baumannii, and 37 kDa from Escherichia coli K12. Comparison of the six porin proteins revealed that, independent of their aggregation state, their amino acid content is similar but not identical. All have double the amount of negatively charged amino acids compared with positively charged amino acids. They have a similar polarity and polarity index. Two of the six tested bacteria do not produce beta-lactamase. These two bacteria were sensitive to the different beta-lactams tested. The other four bacteria were resistant to all or to several beta-lactams.A modified liposome swelling method was used for determining the rate of penetration of charged beta-lactam antibiotics. Zwitterionic beta-lactams were found to penetrate into liposomes at a rate that more or less fits their molecular weight, whether the porins are monomeric or oligomeric. The penetration rates of negatively charged beta-lactams are different for oligomeric and monomeric porins. Negatively charged beta-lactams penetrate through oligomeric porins better than estimated by their molecular weight, whereas monomeric porins are less penetrable to negatively charged beta-lactams than estimated by their molecular weight. The contribution of all types of porins to the susceptibility of bacteria to beta-lactam antibiotics (zwitterionic or negatively charged) is apparently doubtful. The porins may decrease or increase bacterial penetration rates to beta-lactams, and only the existence of a potential beta-lactamase that can destroy the penetrating drug will cause resistance.

Anti-Bacterial Agents↗
PubMed2004

Low density lipoprotein inactivates Vibrio vulnificus cytolysin through the oligomerization of toxin monomer.

Blood lipoprotein has been shown to be an important defense factor against the bacterial infection. Lipoprotein is scavenger of bacterial endotoxin (lipopolysaccharide, LPS). However, there is little evidence showing its protective action against the bacterial exotoxin. We have previously demonstrated that cholesterol inactivates Vibrio vulnificus cytolysin (VVC) through oligomerization of the toxin monomer. The aim of the present study was to investigate the relationship between VVC and low-density lipoprotein cholesterol (LDL-C). LDL induced the oligomerization of VVC in a dose-dependent manner. The oligomerization of VVC monomer with LDL was demonstrated by immunoblotting, which exhibited 200-kDa bands corresponding to a tetramer of the native VVC of relative molecular weight of 51,000. Moreover, LDL inactivated hemolytic activity of VVC in a dose-dependent manner, and this response was not changed by the modifications of LDL (heat denaturation of proteins and peroxidation of phospholipids), suggesting that LDL-C is associated with the defense action against VVC. These results suggest that LDL-C inactivates VVC through the induction of toxin oligomerization.

Animals↗
PubMed2004

Trace metal contamination initiates the apparent auto-aggregation, amyloidosis, and oligomerization of Alzheimer's Abeta peptides.

Nucleation-dependent protein aggregation ("seeding") and amyloid fibril-free formation of soluble SDS-resistant oligomers ("oligomerization") by hydrophobic interaction is an in vitro model thought to propagate beta-amyloid (Abeta) deposition, accumulation, and incur neurotoxicity and synaptotoxicity in Alzheimer's disease (AD), and other amyloid-associated neurodegenerative diseases. However, Abeta is a high-affinity metalloprotein that aggregates in the presence of biometals (zinc, copper, and iron), and neocortical Abeta deposition is abolished by genetic ablation of synaptic zinc in transgenic mice. We now present in vitro evidence that trace (<or=0.8 microM) levels of zinc, copper, and iron, present as common contaminants of laboratory buffers and culture media, are the actual initiators of the classic Abeta1-42-mediated seeding process and Abeta oligomerization. Replicating the experimental conditions of earlier workers, we found that the in vitro precipitation and amyloidosis of Abeta1-40 (20 microM) initiated by Abeta1-42 (2 microM) were abolished by chelation of trace metal contaminants. Further, metal chelation attenuated formation of soluble Abeta oligomers from a cell-free culture medium. These data suggest that protein self-assembly and oligomerization are not spontaneous in this system as previously thought, and that there may be an obligatory role for metal ions in initiating Abeta amyloidosis and oligomerization.

Alzheimer Disease↗
PubMed1991

Regulation of the catalytic activity and oligomeric composition of enzymes in reversed micelles of surfactants in organic solvents.

The phenomenon of regulation of the catalytic activity of enzymes via changing their oligomeric composition in the system of reversed micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in octane was studied using alpha-chymotrypsin (CT) from bovine brain and alkaline phosphatase (AP) from calf intestinal mucosa. The dependences of the enzyme catalytic activity on the AOT hydration degree (Wo = [H2O]/[AOT]), the parameter determining the radius (rc) of the inner cavity of micelles, usually represent the bell-shaped curves. The maximal catalytic activity is observed at such Wo when rc is equal to the size of the enzyme molecule. The position of this maximum strictly correlates with the enzyme oligomeric composition. Thus, in the case of CT this is observed at Wo = 12 when rc is equal to the radius (rp) of the CT globule. In the case of artificially produced conjugate containing six cross-linked CT molecules, this is observed at Wo = 43 when rc is equal to the radius of the sphere surrounding the absolute octahedron composed of six CT globules. The dependence of the catalytic activity of AP on Wo represents a curve with two maxima that are observed when rc is equal to rp of either AP monomer (Wo = 17) or AP dimer (Wo = 25). Ultracentrifugation experiments revealed that variation of Wo causes a change in the oligomeric composition of AP - its transition from monomeric (Wo less than 20) to dimeric form (Wo greater than 20). Hence, the observed maxima correspond to functioning of different oligomeric forms of AP.

Alkaline Phosphatase↗
PubMed1995

Characterization of a soluble, oligomeric HIV-1 gp160 protein as a potential immunogen.

We have assessed the oligomeric structure and antigenic properties of an affinity purified gp160 protein (oligo-gp160) using biosensor technology. Sucrose gradient purification analysis identified the existence of tetrameric, dimeric and monomeric forms of the protein. Reactivity to a broad panel of monoclonal antibodies specific for oligomeric gp160, discontinuous epitopes within monomeric gp120 and several linear epitopes within gp120 (V3) and gp41 was demonstrated. International sera from several countries, where HIV-1 clades A-F are prevalent, including type O from Cameroon, were reactive with oligo-gp160 indicating conserved antigenic epitopes. Enhanced immunologic reactivity per gp160 molecule was obtained with oligo-gp160 as compared to other current HIV-1(IIIB) subunit monomeric envelope gp120/gp160 immunogens suggesting higher HIV-1 envelope protein mimicry. HIV-1 antibodies from sera during acute HIV-1 infection were detectable by oligo-gp160 prior to detection with either a recombinant, monomeric gp120 protein or several commercial HIV-1 screening kits suggesting antibodies sensitive to oligomeric gp160 structure may be present earlier in infection. The oligomeric nature of this gp160 protein preparation and high reactivity with divergent mAbs and HIV-1 sera support the use of this protein as an HIV-1 immunogen.

Antibodies, Monoclonal↗
PubMed1991

Molecular domains involved in oligomerization of the Friend murine leukemia virus envelope glycoprotein.

The oligomeric structure of the Friend murine leukemia virus envelope glycoprotein has been investigated using crosslinking reagents and sucrose density gradient centrifugation. The results obtained provide evidence that both the precursor and the processed molecules are oligomeric and probably form tetramers. Pulse-chase analyses indicate that assembly occurs sequentially, within 30 min of protein synthesis and prior to cleavage of the precursor. Studies using chimeric envelope glycoproteins and deletion mutants indicate that the transmembrane and cytoplasmic domains are not essential for the formation of oligomers. Evidence is also presented that the SU subunit remains in an oligomeric form following disassociation from the TM subunit. Oligomeric envelope glycoprotein complexes linked by intermolecular disulfide bonds were also observed under certain conditions. Mink cell focus-forming virus envelope glycoprotein constructs lacking the transmembrane domain or both the transmembrane and the cytoplasmic domains formed intermolecular disulfide bonds more readily than the full-length molecule, suggesting that these regions are likely to make a contribution to the conformation of the glycoprotein. These data indicate that there are several points of interaction between retrovirus envelope glycoprotein monomers which contribute to assembly of the oligomer and that contacts within the ectodomain appear to be of critical importance.

Amino Acid Sequence↗
PubMed1982

Membrane insertion and oligomeric assembly of HLA-DR histocompatibility antigens.

HLA-DR histocompatibility antigens are assembled in the endoplasmic reticulum. This assembly has been studied in vitro and in vivo. Three polypeptides are involved in forming the oligomeric structure of HLA-DR antigens, DR alpha chains (molecular weight 35,000), DR beta chains (molecular weight 29,000) and DR gamma chains (molecular weight 33,000). They are cotranslationally inserted into the membrane of the endoplasmic reticulum, and all span the membrane. The size of the cytoplasmic portion of DR alpha and DR beta is about 500- 1000 daltons, whereas that of the DR gamma chain is about 3000 daltons. Oligomeric assembly of DR alpha, DR beta and DR gamma chains occurs shortly after their synthesis in the endoplasmic reticulum. DR gamma chains are synthesized in excess of DR alpha and DR beta chains, and hence in the endoplasmic reticulum they are found either in a complex with DR alpha and DR beta or in a free form. Free DR gamma chains remain in the endoplasmic reticulum, whereas DR gamma chains present in the oligomeric complex with DR alpha and DR beta undergo intracellular transport. Their molecular weight increases during transport, probably because of the addition of complex sugars in the Golgi complex. This is followed by the detachment of DR gamma chains from the oligomeric complex and the appearance of DR alpha and DR beta chains on the cell surface. Whether any DR gamma chains appear on the cell surface is uncertain.

Biological Transport↗
PubMed1994

Prolyl isomerase requirement for the expression of functional homo-oligomeric ligand-gated ion channels.

Ligand-gated ion channel subunits show a striking abundance of highly conserved proline residues. We, therefore, tested the hypothesis that peptidyl-prolyl isomerases may be involved in the maturation of these channels. Cyclosporin A, a selective blocker of a ubiquitous isomerase cyclophilin, reduced the surface expression in Xenopus oocytes of functional homo-oligomeric receptors containing nicotinic acetylcholine receptor subunit alpha 7 without blocking alpha 7 polypeptide synthesis. This effect could be generalized to the homo-oligomeric 5-hydroxytryptamine type 3 receptor but not to the hetero-oligomeric muscle nicotinic receptor. An alpha 7 receptor could be rescued from cyclosporin A blockade by coexpressed muscle non-alpha subunits. The effect of cyclosporin A was reversed by overexpression of exogenous rat brain cyclophilin. These findings indicate that cyclophilins may play a critical role in the maturation of homo-oligomeric receptors, acting directly or indirectly as prolyl isomerases or as molecular chaperones.

Amino Acid Isomerases↗
PubMed2005

CaMK-II oligomerization potential determined using CFP/YFP FRET.

Members of the Ca(2+)/calmodulin-dependent protein kinase II (CaMK-II) family are encoded throughout the animal kingdom by up to four genes (alpha, beta, gamma, and delta). Over three dozen known CaMK-II splice variants assemble into approximately 12-subunit oligomers with catalytic domains facing out from a central core. In this study, the catalytic domain of alpha, beta, and delta CaMK-IIs was replaced with cyan (CFP) or yellow fluorescent protein (YFP) for fluorescence resonance energy transfer (FRET) studies. FRET, when normalized to total CFP and YFP, reproducibly yielded values which reflected oligomerization preference, inter-subunit spacing, and localization. FRET occurred when individual CFP and YFP-linked CaMK-IIs were co-expressed, but not when they were expressed separately and then mixed. All hetero-oligomers exhibited FRET values that were averages of their homo-oligomeric parents, indicating no oligomeric preference or restriction. FRET for CaMK-II homo-oligomers was inversely proportional to the variable region length. FPs were monomerized (Leu221 to Lys221) for this study, thus eliminating any potential artifact caused by FP-CaMK-II aggregates. Our results indicate that alpha, beta, and delta CaMK-IIs can freely hetero-oligomerize and that increased variable region lengths place amino termini further apart, potentially influencing the rate of inter-subunit autophosphorylation.

Amino Acid Sequence↗
PubMed2004

Oligomeric Aip2p/Dld2p modifies the protein conformation of both properly folded and misfolded substrates in vitro.

Oligomeric actin-interacting protein 2 (Aip2p) [Nat. Struct. Biol. 2 (1995) 28]/D-lactate dehydrogenase protein 2 (Dld2p) [Yeast 15 (1999) 1377, Biochem. Biophys. Res. Commun. 295 (2002) 910] exhibits the unique grapple-like structure with an ATP-dependent opening [Biochem. Biophys. Res. Commun. 320 (2004) 1271], which is required for the F-actin conformation modifying activity in vitro and in vivo [Biochem. Biophys. Res. Commun. 319 (2004) 78]. To further investigate the molecular nature of oligomeric Aip2p/Dld2p, the substrate specificity of its binding and protein conformation modifying activity was examined. In the presence of 1mM ATP or AMP-PNP, oligomeric Aip2p/Dld2p bound to all substrates so far examined, and modified the conformation of actin, DNase I, the mature form of invertase, prepro-alpha-factor, pro-alpha-factor, and mitochondrial superoxide dismutase, as determined by the trypsin susceptibility assay. Of note, the activity could modify even the conformation of pathogenic highly aggregated polypeptides, such as recombinant prion protein in beta-sheet form, alpha-synuclein, and amyloid beta (1-42) in the presence of ATP. The in vivo protein conformation modifying activity, however, depends on the growth stage; the most significant substrate modification activity was observed in yeast cells at the log phase, suggesting the presence of a cofactor/s in yeast cells, where F-actin is supposed to be a major target in vivo. These data further support our previous notion that the oligomeric Aip2p/Dld2p may belong to an unusual class of molecular chaperones [Biochem. Biophys. Res. Commun. 320 (2004) 1271], which can target both properly folded and misfolded proteins in an ATP-dependent manner in vitro.

Actins↗