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1.
The binding protein (BiP; a member of the heat-shock 70 family) is a major chaperone of the endoplasmic reticulum (ER). Interactions with BiP are believed to inhibit unproductive aggregation of newly synthesized secretory proteins during folding and assembly. In vitro, BiP has a preference for peptide sequences enriched in hydrophobic amino acids, which are expected to be exposed only in folding and assembly intermediates or in defective proteins. However, direct information regarding sequences recognized in vivo by BiP on real proteins is very limited. We have shown previously that newly synthesized monomers of the homotrimeric storage protein phaseolin associate with BiP and that phaseolin trimerization in the ER abolishes such interactions. Using different phaseolin constructs and green fluorescent protein (GFP) fusion proteins, we show here that one of the two alpha-helical regions of polypeptide contact in phaseolin trimers (35 amino acids located close to the C terminus and containing three potential BiP binding sites) effectively promotes BiP association with phaseolin and with secretory GFP fusions expressed in transgenic tobacco or in transfected protoplasts. We also show that overexpressed BiP transiently sequesters phaseolin polypeptides. We conclude that one of the regions of monomer contact is a BiP binding determinant and suggest that during the synthesis of phaseolin, the association with BiP and trimer formation are competing events. Finally, we show that the other, internal region of contact between monomers is necessary for phaseolin assembly in vivo and contains one potential BiP binding site.  相似文献   

2.
The mass-driven assembly of spectrin dimers to form tetramers involves two equal head-to-head alpha-beta associations and requires at least 30 degrees C for interconversion to occur readily. In this paper, the properties of tetramer formation were investigated using two complementary univalent peptides (the alpha I domain and beta monomers). Since the alpha I domain lacks an essential nucleation site required for side-to-side (lateral) heterodimer assembly [Speicher et al. (1992) J. Biol. Chem. 267, 14775-14782], these two peptides can only assemble head-to-head at a single site. This head-to-head assembly readily occurs at lower temperatures, indicating the temperature barrier for dimer-tetramer interconversion is caused by a conformational constraint of the dimer. This constraint, a closed hairpin loop, is released when the laterally associated partner is removed. The univalent alpha I-beta binding affinity at 37 degrees C (Ka = 1.4 x 10(5) M-1) is similar to the dimer-tetramer association constant at the same temperature. As the temperature is decreased from 37 to 0 degrees C, the alpha I-beta binding affinity increases about 32-fold. In contrast with head-to-head associations involving dimers, the second-order rate constants of two complementary univalent peptides (i.e., alpha I and beta) are dramatically higher, and the estimated activation energy (about 50 kJ mol-1) is about 5-fold lower. An open dimer conformation is an obligatory high-energy intermediate required for dimer-tetramer interconversion, and opening the dimer hairpin loop contributes about 190 kJ mol-1 to the activation energy for tetramer association.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

3.
Protein aggregation is a multi-step process that requires sequential structural transitions of monomers during their incorporation into oligomers. Such process involves the formation of various intermediate stages in protein structures. Seed-nucleation mediated oligomerization is observed in many aggregation-prone proteins. Understanding of the protein seed's structural features and mechanisms of its transition-state formation are important for knowing the details of post-nucleation aggregation process. We have identified the metastable states in the seeds of the Ubiquitin associated (UBA) domain of Huntingtin Interacting Protein K (HYPK). This is studied by monitoring the events of dynamic transitions of metastable seeds to aggregates or monomers through microscopy, biophysical and computational techniques. HYPK-UBA seeds can exist in specific metastable state(s) that show transition from closed to open conformations, thereby reorienting the helix associated hydrophobic patches to cause its self-aggregation. Metastable seeds show inter-seed exchange of monomers through simultaneous dissociation-association phenomenon. Monomer release from metastable seeds can cause the dissolution of the aggregates. Like metastable monomers, metastable seeds also show reduction in their secondary structure by altering the molecular contacts and solvent accessible hydrophobic surfaces. Induction of metastable seeds from the ground-state is a slow thermodynamic process and it results from excitable perturbations. Conclusively, we propose the concept that the thermodynamic induction of metastable states in HYPK-UBA seed potentiates the molecule to switch its conformations that increases the protein's self-aggregation by the mechanism of hydrophobic patch collapse, while also releasing the monomers from oligomeric seeds due to structural instability.  相似文献   

4.
Septins constitute a family of guanine nucleotide-binding proteins that were first discovered in the yeast Saccharomyces cerevisiae but are also present in many other eukaryotes. In yeast they congregate at the bud neck and are required for cell division. Their function in metazoan cells is uncertain, but they have been implicated in exocytosis and cytokinesis. Septins have been purified from cells as hetero-oligomeric filaments, but their mechanism of assembly is unknown. Further studies have been limited by the difficulty in expressing functional septin proteins in bacteria. We now show that stable, soluble septin heterodimers can be produced by co-expression from bicistronic vectors in bacteria and that the co-expression of three septins results in their assembly into filaments. Pre-assembled dimers and trimers bind guanine nucleotide and show a slow GTPase activity. The assembly of a heterodimer from monomers in vitro is accompanied by GTP hydrolysis. Borg3, a downstream effector of the Cdc42 GTPase, binds specifically to a septin heterodimer composed of Sept6 and Sept7 and to the Sept2/6/7 trimer, but not to septin monomers or to other heterodimers. Septins associate through their C-terminal coiled-coil domains, and Borg3 appears to recognize the interface between these domains in Sept6 and Sept7.  相似文献   

5.
Using comparative modeling, we have generated structural models of 475 α and β tubulins. Using these models, we observed a global, structural similarity between the tubulin isotypes. However, a number of subtle differences in the isotypes physical properties, including net electric charges, solvent accessible surface areas, and electric dipole moments were also apparent. In order to examine the roles that these properties may play in microtubule (MT) assembly and stability, we have created a model to evaluate the dipole–dipole interaction energies of varying MT lattice conformations, using human tubulin isotypes as particularly important examples. We conclude that the dipole moments of each tubulin isotype may influence their functional characteristics within the cell, resulting in differences for MT assembly kinetics and stability.  相似文献   

6.
Data sets of 362 structurally nonredundant protein-protein interfaces and of 57 symmetry-related oligomeric interfaces have been used to explore whether the hydrophobic effect that guides protein folding is also the main driving force for protein-protein associations. The buried nonpolar surface area has been used to measure the hydrophobic effect. Our analysis indicates that, although the hydrophobic effect plays a dominant role in protein-protein binding, it is not as strong as that observed in the interior of protein monomers. Comparison of interiors of the monomers with those of the interfaces reveals that, in general, the hydrophobic amino acids are more frequent in the interior of the monomers than in the interior of the protein-protein interfaces. On the other hand, a higher proportion of charged and polar residues are buried at the interfaces, suggesting that hydrogen bonds and ion pairs contribute more to the stability of protein binding than to that of protein folding. Moreover, comparison of the interior of the interfaces to protein surfaces indicates that the interfaces are poorer in polar/charged than the surfaces and are richer in hydrophobic residues. The interior of the interfaces appears to constitute a compromise between the stabilization contributed by the hydrophobic effect on the one hand and avoiding patches on the protein surfaces that are too hydrophobic on the other. Such patches would be unfavorable for the unassociated monomers in solution. We conclude that, although the types of interactions are similar between protein-protein interfaces and single-chain proteins overall, the contribution of the hydrophobic effect to protein-protein associations is not as strong as to protein folding. This implies that packing patterns and interatom, or interresidue, pairwise potential functions, derived from monomers, are not ideally suited to predicting and assessing ligand associations or design. These would perform adequately only in cases where the hydrophobic effect at the binding site is substantial.  相似文献   

7.
Bacterial cytoskeletal protein FtsZ assembles in a head-to-tail manner, forming dynamic filaments that are essential for cell division. Here, we study their dynamics using unbiased atomistic molecular simulations from representative filament crystal structures. In agreement with experimental data, we find different filament curvatures that are supported by a nucleotide-regulated hinge motion between consecutive FtsZ monomers. Whereas GTP-FtsZ filaments bend and twist in a preferred orientation, thereby burying the nucleotide, the differently curved GDP-FtsZ filaments exhibit a heterogeneous distribution of open and closed interfaces between monomers. We identify a coordinated Mg2+ ion as the key structural element in closing the nucleotide site and stabilizing GTP filaments, whereas the loss of the contacts with loop T7 from the next monomer in GDP filaments leads to open interfaces that are more prone to depolymerization. We monitored the FtsZ monomer assembly switch, which involves opening/closing of the cleft between the C-terminal domain and the H7 helix, and observed the relaxation of isolated and filament minus-end monomers into the closed-cleft inactive conformation. This result validates the proposed switch between the low-affinity monomeric closed-cleft conformation and the active open-cleft FtsZ conformation within filaments. Finally, we observed how the antibiotic PC190723 suppresses the disassembly switch and allosterically induces closure of the intermonomer interfaces, thus stabilizing the filament. Our studies provide detailed structural and dynamic insights into modulation of both the intrinsic curvature of the FtsZ filaments and the molecular switch coupled to the high-affinity end-wise association of FtsZ monomers.  相似文献   

8.
Amyloid fibrils, which are polymeric assemblies of protein molecules, have been intensively studied on a structural level, yet due to complications such as the disorder within the molecules, several aspects of their structure remain mysterious. Similarly, the kinetics of assembly are not well understood. Here we investigate the electric dipole moment of beta-lactoglobulin fibrils, a model amyloid fibril system, by applying the technique of transient electric birefringence. This moment appears to be large, and comparable to the total moment of the constituent protein monomers if they were joined in a chain, head-to-tail, without changing conformation, suggesting an ordered joining of monomers in the fibril. Such an ordered assembly may have implications for the assembly mechanism of beta-lactoglobulin fibrils in particular, and amyloid fibrils in general.  相似文献   

9.
Homotetrameric proteins can assemble by several different pathways, but have only been observed to use one, in which two monomers associate to form a homodimer, and then two homodimers associate to form a homotetramer. To determine why this pathway should be so uniformly dominant, we have modeled the kinetics of tetramerization for the possible pathways as a function of the rate constants for each step. We have found that competition with the other pathways, in which homotetramers can be formed either by the association of two different types of homodimers or by the successive addition of monomers to homodimers and homotrimers, can cause substantial amounts of protein to be trapped as intermediates of the assembly pathway. We propose that this could lead to undesirable consequences for an organism, and that selective pressure may have caused homotetrameric proteins to evolve to assemble by a single pathway.  相似文献   

10.
Bacterial cytoskeletal protein FtsZ assembles in a head-to-tail manner, forming dynamic filaments that are essential for cell division. Here, we study their dynamics using unbiased atomistic molecular simulations from representative filament crystal structures. In agreement with experimental data, we find different filament curvatures that are supported by a nucleotide-regulated hinge motion between consecutive FtsZ monomers. Whereas GTP-FtsZ filaments bend and twist in a preferred orientation, thereby burying the nucleotide, the differently curved GDP-FtsZ filaments exhibit a heterogeneous distribution of open and closed interfaces between monomers. We identify a coordinated Mg2+ ion as the key structural element in closing the nucleotide site and stabilizing GTP filaments, whereas the loss of the contacts with loop T7 from the next monomer in GDP filaments leads to open interfaces that are more prone to depolymerization. We monitored the FtsZ monomer assembly switch, which involves opening/closing of the cleft between the C-terminal domain and the H7 helix, and observed the relaxation of isolated and filament minus-end monomers into the closed-cleft inactive conformation. This result validates the proposed switch between the low-affinity monomeric closed-cleft conformation and the active open-cleft FtsZ conformation within filaments. Finally, we observed how the antibiotic PC190723 suppresses the disassembly switch and allosterically induces closure of the intermonomer interfaces, thus stabilizing the filament. Our studies provide detailed structural and dynamic insights into modulation of both the intrinsic curvature of the FtsZ filaments and the molecular switch coupled to the high-affinity end-wise association of FtsZ monomers.  相似文献   

11.
The Yersinia enterocolitica phage shock protein (Psp) stress response is essential for virulence and for survival during the mislocalization of outer membrane secretin proteins. The cytoplasmic membrane proteins PspB and PspC are critical components involved in regulating psp gene expression and in facilitating tolerance to secretin-induced stress. Interactions between PspB and PspC monomers might be important for their functions and for PspC stability. However, little is known about these interactions and there are conflicting reports about the ability of PspC to dimerize. To address this, we have used a combination of independent approaches to systematically analyze the ability of PspB and PspC to form dimers in vivo. Formaldehyde cross-linking of the endogenous chromosomally encoded proteins in Y. enterocolitica revealed discrete complexes corresponding in size to PspB-PspB, PspC-PspC, and PspB-PspC. Bacterial two-hybrid analysis corroborated these protein associations, but an important limitation of the two-hybrid approach was uncovered for PspB. A series of PspB and PspC proteins with unique cysteine substitutions at various positions was constructed. In vivo disulfide cross-linking experiments with these proteins further supported close association between PspB and PspC monomers. Detailed cysteine substitution analysis of predicted leucine zipper-like amphipathic helices in both PspB and PspC suggested that their hydrophobic faces could form homodimerization interfaces.  相似文献   

12.
Small-angle X-ray scattering experiments were carried out for the maltose-, glucose/galactose- and ribose-binding proteins of Gram negative bacteria. All were shown to be monomers that decrease in radius of gyration on ligand binding.The results obtained for the maltose-binding protein agree well with crystal structures of the closed, ligand-bound, and open, ligand-free protein, suggesting that these are indeed the primary forms in solution. The closed form is stabilized by protein – sugar interactions, while the open conformation is stabilized by close contacts between the two domains. Since it is the proper spacial relationship of the domains in the closed form that is most important for interaction with chemotaxis and transport partners, the stabilization of the open form would help keep ligand-free molecules from interfering in function.The scattering results also provide evidence that a large conformational change takes place in association with ligand binding to the glucose/galactose- and ribose-binding proteins, and that the two changes are similar. Modeling suggests that the open forms resemble those found in the related leucine and leucine/isoleucine/valine-binding proteins, but are different from those observed for the maltose-binding protein and the related purine repressor.  相似文献   

13.
Surface, subunit interfaces and interior of oligomeric proteins   总被引:41,自引:0,他引:41  
The solvent-accessible surface area (As) of 23 oligomeric proteins is calculated using atomic co-ordinates from high-resolution and well-refined crystal structures. As is correlated with the protein molecular weight, and a power law predicts its value to within 5% on average. The accessible surface of the average oligomer is similar to that of monomeric proteins in its hydropathy and amino acid composition. The distribution of the 20 amino acid types between the protein surface and its interior is also the same as in monomers. Interfaces, i.e. surfaces involved in subunit contacts, differ from the rest of the subunit surface. They are enriched in hydrophobic side-chains, yet they contain a number of charged groups, especially from Arg residues, which are the most abundant residues at interfaces except for Leu. Buried Arg residues are involved in H-bonds between subunits. We counted H-bonds at interfaces and found that several have none, others have one H-bond per 200 A2 of interface area on average (1 A = 0.1 nm). A majority of interface H-bonds involve charged donor or acceptor groups, which should make their contribution to the free energy of dissociation significant, even when they are few. The smaller interfaces cover about 700 A2 of the subunit surface. The larger ones cover 3000 to 10,000 A2, up to 40% of the subunit surface area in catalase. The lower value corresponds to an estimate of the accessible surface area loss required for stabilizing subunit association through the hydrophobic effect alone. Oligomers with small interfaces have globular subunits with accessible surface areas similar to those of monomeric proteins. We suggest that these oligomers assemble from preformed monomers with little change in conformation. In oligomers with large interfaces, isolated subunits should be unstable given their excessively large accessible surface, and assembly is expected to require major structural changes.  相似文献   

14.
The timothy grass pollen allergen Phl p 7 assembles most of the IgE epitopes of a novel family of 2 EF-hand calcium-binding proteins and therefore represents a diagnostic marker allergen and vaccine candidate for immunotherapy. Here we report the first three-dimensional structure of a representative of the 2 EF-hand allergen family, Phl p 7, in the calcium-bound form. The protein occurs as a novel dimer assembly with unique features: in contrast to well known EF-hand proteins such as calmodulin, parvalbumin or the S100 proteins, Phl p 7 adopts an extended conformation. Two protein monomers assemble in a head-to-tail arrangement with domain-swapped EF-hand pairing. The intertwined dimer adopts a barrel-like structure with an extended hydrophobic cavity providing a ligand-binding site. Calcium binding acts as a conformational switch between an open and a closed dimeric form of Phl p 7. These findings are interesting in the context of lipid- and calcium-dependent pollen tube growth. Furthermore, the structure of Phl p 7 allows for the rational development of vaccine strategies for treatment of sensitized allergic patients.  相似文献   

15.
Two folate binding proteins are present in human milk; one of 27 kDa is a cleavage product of the other one (100 kDa) which possesses a hydrophobic membrane anchor. A drastic change of radioligand binding characteristics and appearance of aggregated weak-radioligand affinity forms on gel filtration occurred at low concentrations of both proteins in the absence of Triton X-100 or other amphiphatic substances, e.g. cetyltrimethylammonium and phospholipids. These findings are consistent with a model predicting association between unliganded and liganded monomers resulting in weak-ligand affinity dimers. Amphiphatic substances form micelles and lipid bilayers which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers (monomers become hydrophilic in the liganded state) thereby preventing association between these monomeric forms prevailing at low concentrations of the protein. Bio-Gel P-300 chromatography of the 27 kDa protein revealed a pronounced polymerization tendency, which diminished with decreasing protein concentrations, however, not in the presence of cetyltrimethylammonium. The data could have some bearings on observations indicating that naturally occurring amphiphatic substances, cholesterol and phospholipids, are necessary for the important clustering of membrane folate receptors.  相似文献   

16.
Ubiquitin (Ub) is a small protein highly conserved among eukaryotes and involved in practically all aspects of eukaryotic cell biology. Polymeric chains assembled from covalently-linked Ub monomers function as molecular signals in the regulation of a host of cellular processes. Our previous studies have shown that the predominant state of Lys48-linked di- and tetra-Ub chains at near-physiological conditions is a closed conformation, in which the Ub-Ub interface is formed by the hydrophobic surface residues of the adjacent Ub units. Because these very residues are involved in (poly)Ub interactions with the majority of Ub-binding proteins, their sequestration at the Ub-Ub interface renders the closed conformation of polyUb binding incompetent. Thus the existence of open conformation(s) and the interdomain motions opening and closing the Ub-Ub interface is critical for the recognition of Lys48-linked polyUb by its receptors. Knowledge of the conformational properties of a polyUb signal is essential for our understanding of its specific recognition by various Ub-receptors. Despite their functional importance, open states of Lys48-linked chains are poorly characterized. Here we report a crystal structure of the open state of Lys48-linked di-Ub. Moreover, using NMR, we examined interactions of the open state of this chain (at pH4.5) with a Lys48-linkage-selective receptor, the UBA2 domain of a shuttle protein hHR23a. Our results show that di-Ub binds UBA2 in the same mode and with comparable affinity as the closed state. Our data suggest a mechanism for polyUb signal recognition, whereby Ub-binding proteins select specific conformations out of the available ensemble of polyUb chain conformations. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.  相似文献   

17.
Exposure of a phycocyanin-phycoerythrocyanin mixture extracted from Anabaena variabilis to sodium acetate, pH 3.8, ionic strength of 0.1, results in dissociation of the phycoerythrocyanin's beta subunit from its alpha subunit. The alpha subunit obtained by this method has a strong absorption transition at 508 nm. This transition is a consequence of the subunit's specific conformation, rather than of a new chromophore. The behavior of the phycocyanin-phycoerythrocyanin mixture in acetate buffers of variable compositions suggests that interactions which involve carboxylic amino acid residues play an important role, along with hydrophobic associations, in the association of phycoerythrocyanin subunits into monomers (alpha beta) and between this protein and phycocyanin. This work also indicates that the linkage between alpha and beta subunits of phycoerythrocyanin is labile and may be weaker than the association of these subunits with phycocyanin under acidic conditions.  相似文献   

18.
Speed and processivity of replicative DNA polymerases can be enhanced via coupling to a sliding clamp. Due to the closed ring shape of the clamp, a clamp loader protein, belonging to the AAA+ class of ATPases, needs to open the ring-shaped clamp before loading it to DNA. Here, we developed real-time fluorescence assays to study the clamp (PCNA) and the clamp loader (RFC) from the mesophilic archaeon Methanosarcina acetivorans. Unexpectedly, we discovered that RFC can assemble a PCNA ring from monomers in solution. A motion-based DNA polymerization assay showed that the PCNA assembled by RFC is functional. This PCNA assembly activity required the ATP-bound conformation of RFC. Our work demonstrates a reverse-chaperoning activity for an AAA+ protein that can act as a template for the assembly of another protein complex.  相似文献   

19.
A potential barrier to the development of genetically targeted adenovirus (Ad) vectors for cell-specific delivery of gene therapeutics lies in the fact that several types of targeting protein ligands require posttranslational modifications, such as the formation of disulfide bonds, which are not available to Ad capsid proteins due to their nuclear localization during assembly of the virion. To overcome this problem, we developed a new targeting strategy, which combines genetic modifications of the Ad capsid with a protein bridge approach, resulting in a vector-ligand targeting complex. The components of the complex associate by virtue of genetic modifications to both the Ad capsid and the targeting ligand. One component of this mechanism of association, the Fc-binding domain of Staphylococcus aureus protein A, is genetically incorporated into the Ad fiber protein. The ligand is comprised of a targeting component fused with the Fc domain of immunoglobulin, which serves as a docking moiety to bind to these genetically modified fibers during the formation of the Ad-ligand complex. The modular design of the ligand solves the problem of structural and biosynthetic compatibility with the Ad and thus facilitates targeting of the vector to a variety of cellular receptors. Our study shows that targeting ligands incorporating the Fc domain and either an anti-CD40 single-chain antibody or CD40L form stable complexes with protein A-modified Ad vectors, resulting in significant augmentation of gene delivery to CD40-positive target cells. Since this gene transfer is independent of the expression of the native Ad5 receptor by the target cells, this strategy results in the derivation of truly targeted Ad vectors suitable for tissue-specific gene therapy.  相似文献   

20.
The effects of the hydrophobicity and the distribution of hydrophobic residues on the surfaces of some designed alpha-helical transmembrane peptides (acetyl-K2-L(m)-A(n)-K2-amide, where m + n = 24) on their solution behavior and interactions with phospholipids were examined. We find that although these peptides exhibit strong alpha-helix forming propensities in water, membrane-mimetic media, and lipid model membranes, the stability of the helices decreases as the Leu content decreases. Also, their binding to reversed phase high-performance liquid chromatography columns is largely determined by their hydrophobicity and generally decreases with decreases in the Leu/Ala ratio. However, the retention of these peptides by such columns is also affected by the distribution of hydrophobic residues on their helical surfaces, being further enhanced when peptide helical hydrophobic moments are increased by clustering hydrophobic residues on one side of the helix. This clustering of hydrophobic residues also increases peptide propensity for self-aggregation in aqueous media and enhances partitioning of the peptide into lipid bilayer membranes. We also find that the peptides LA3LA2 [acetyl-K2-(LAAALAA)3LAA-K2-amide] and particularly LA6 [acetyl-K2-(LAAAAAA)3LAA-K2-amide] associate less strongly with and perturb the thermotropic phase behavior of phosphatidylcholine bilayers much less than peptides with higher L/A ratios. These results are consistent with free energies calculated for the partitioning of these peptides between water and phospholipid bilayers, which suggest that LA3LA2 has an equal tendency to partition into water and into the hydrophobic core of phospholipid model membranes, whereas LA6 should strongly prefer the aqueous phase. We conclude that for alpha-helical peptides of this type, Leu/Ala ratios of greater than 7/17 are required for stable transmembrane associations with phospholipid bilayers.  相似文献   

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