共查询到20条相似文献,搜索用时 15 毫秒
1.
Dynamics and function of proteins are governed by the structural and energetic properties of the different states they adopt and the barriers separating them. In earlier work, native-state triplet–triplet energy transfer (TTET) on the villin headpiece subdomain (HP35) revealed an equilibrium between a locked native state and an unlocked native state, which are structurally similar but have different dynamic properties. The locked state is restricted to low amplitude motions, whereas the unlocked state shows increased conformational flexibility and undergoes local unfolding reactions. This classified the unlocked state as a dry molten globule (DMG), which was proposed to represent an expanded native state with loosened side-chain interactions and a solvent-shielded core. To test whether the unlocked state of HP35 is actually expanded compared to the locked state, we performed high-pressure TTET measurements. Increasing pressure shifts the equilibrium from the locked toward the unlocked state, with a small negative reaction volume for unlocking (ΔV0 = − 1.6 ± 0.5 cm3/mol). Therefore, rather than being expanded, the unlocked state represents an alternatively packed, compact state, demonstrating that native proteins can exist in several compact folded states, an observation with implications for protein function. The transition state for unlocking/locking, in contrast, has a largely increased volume relative to the locked and unlocked state, with respective activation volumes of 7.1 ± 0.4 cm3/mol and 8.7 ± 0.9 cm3/mol, indicating an expansion of the protein during the locking/unlocking transition. The presented results demonstrate the existence of both compact, low-energy and expanded, high-energy DMGs, prompting a broader definition of this state. 相似文献
2.
Ryo P. Honda Kei-ichi Yamaguchi Kazuo Kuwata 《The Journal of biological chemistry》2014,289(44):30355-30363
The conversion of a cellular prion protein (PrPC) to its pathogenic isoform (PrPSc) is a critical event in the pathogenesis of prion diseases. Pathogenic conversion is usually associated with the oligomerization process; therefore, the conformational characteristics of the pre-oligomer state may provide insights into the conversion process. Previous studies indicate that PrPC is prone to oligomer formation at low pH, but the conformation of the pre-oligomer state remains unknown. In this study, we systematically analyzed the acid-induced conformational changes of PrPC and discovered a unique acid-induced molten globule state at pH 2.0 termed the “A-state.” We characterized the structure of the A-state using far/near-UV CD, 1-anilino-8-naphthalene sulfonate fluorescence, size exclusion chromatography, and NMR. Deuterium exchange experiments with NMR detection revealed its first unique structure ever reported thus far; i.e. the Strand 1-Helix 1-Strand 2 segment at the N terminus was preferentially unfolded, whereas the Helix 2-Helix 3 segment at the C terminus remained marginally stable. This conformational change could be triggered by the protonation of Asp144, Asp147, and Glu196, followed by disruption of key salt bridges in PrPC. Moreover, the initial population of the A-state at low pH (pH 2.0–5.0) was well correlated with the rate of the β-rich oligomer formation, suggesting that the A-state is the pre-oligomer state. Thus, the specific conformation of the A-state would provide crucial insights into the mechanisms of oligomerization and further pathogenic conversion as well as facilitating the design of novel medical chaperones for treating prion diseases. 相似文献
3.
Saha R Dasgupta S Banerjee R Mitra-Bhattacharyya A Söll D Basu G Roy S 《Biochemistry》2012,51(22):4429-4437
Molten globule and other disordered states of proteins are now known to play important roles in many cellular processes. From equilibrium unfolding studies of two paralogous proteins and their variants, glutaminyl-tRNA synthetase (GlnRS) and two of its variants [glutamyl-tRNA synthetase (GluRS) and its isolated domains, and a GluRS-GlnRS chimera], we demonstrate that only GlnRS forms a molten globule-like intermediate at low urea concentrations. We demonstrated that a loop in the GlnRS C-terminal anticodon binding domain that promotes communication with the N-terminal domain and indirectly modulates amino acid binding is also responsible for stabilization of the molten globule state. This loop was inserted into GluRS in the eukaryotic branch after the archaea-eukarya split, right around the time when GlnRS evolved. Because of the structural and functional importance of the loop, it is proposed that the insertion of the loop into a putative ancestral GluRS in eukaryotes produced a catalytically active molten globule state. Because of their enhanced dynamic nature, catalytically active molten globules are likely to possess broad substrate specificity. It is further proposed that the putative broader substrate specificity allowed the catalytically active molten globule to accept glutamine in addition to glutamic acid, leading to the evolution of GlnRS. 相似文献
4.
Marina D. Kirkitadze Paul N. Barlow Nicholas C. Price Sharon M. Kelly Christopher J. Boutell Frazer J. Rixon David A. McClelland 《Journal of virology》1998,72(12):10066-10072
Two proteins, VP19C (50,260 Da) and VP23 (34,268 Da), make up the triplexes which connect adjacent hexons and pentons in the herpes simplex virus type 1 capsid. VP23 was expressed in Escherichia coli and purified to homogeneity by Ni-agarose affinity chromatography. In vitro capsid assembly experiments demonstrated that the purified protein was functionally active. Its physical status was examined by differential scanning calorimetry, ultracentrifugation, size exclusion chromatography, circular dichroism, fluorescence spectroscopy, and 8-anilino-1-naphthalene sulfonate binding studies. These studies established that the bacterially expressed VP23 exhibits properties consistent with its being in a partially folded, molten globule state. We propose that the molten globule represents a functionally relevant intermediate which is necessary to allow VP23 to undergo interaction with VP19C in the process of capsid assembly. 相似文献
5.
核小体是构成染色质的基本结构单位,它使得染色质中DNA、RNA和蛋白质组织成为一种致密的结构形式。其中组蛋白尾部区域常常发生形式多样的翻译后修饰。这包括乙酰化、磷酸化、甲基化、泛蛋白化以及ADP核糖基化。存在于一个或更多的组蛋白尾部的相应的修饰,形成了一种“组蛋白密码”,许多的蛋白质因子对其加以识别,从而引发一系列的下游过程。 相似文献
6.
Guanidine hydrochloride-induced unfolding of a carbonic anhydrase molten globule was studied by high-resolution nuclear magnetic resonance spectroscopy. The study resulted in estimation of the number of water and denaturant molecules bound to the molten globule at various denaturant concentrations in solution. When compared with the data on unfolding of native carbonic anhydrase, these estimates indicate that the unfolding is underlain by an increased local concentration of the denaturant near the protein molecule, which results from the increased ratio between guanidine hydrochloride-bound and protein-bound waters. 相似文献
7.
Hyperglycemic condition i.e. an increase in blood glucose concentration has been linked to bring about structural alterations in the native state of proteins. Glucose concentrations of 50 and 100 mM in vitro, which correspond to hyperglycemic condition, were tested to investigate their effect on lysozyme native structure. Incubating enzyme with 50 and 100 mM glucose for a period of 7 days, an intermediate state on day 4 and 3 was observed, respectively. The presence of intermediate state was characterized by a 22 % increase in the intrinsic fluorescence intensity with a red shift of 20 nm compared to the native state, a 5 % increase in ANS-fluorescence intensity relative to the native due to the surfacing of hydrophobic clusters and a sharp decrease in near-UV CD signal at around 284 and 291 nm. The state retains substantial native-like secondary structure. This partially unfolded intermediate state can be referred as ‘molten globule’, which finally tends to aggregate on day 6 and 4 with 50 and 100 mM glucose concentration, respectively, as a result of cross-linking between lysozyme molecules. The aggregates were confirmed by the presence of β-sheet structure as depicted by far-UV CD, an increase in ThT fluorescence as well as the fibrillar morphology shown by SEM. Moreover, advanced glycation end products were also accompanied as the emission peak was observed at 460 and 470 nm corresponding to the formation of pentosidine and malonaldehyde, respectively. 相似文献
8.
Hamidur Rahaman Md. Khurshid Alam Khan Md. Imtaiyaz Hassan Asimul Islam Ali Akbar Moosavi-Movahedi Faizan Ahmad 《PloS one》2015,10(4)
While many proteins are recognized to undergo folding via intermediate(s), the heterogeneity of equilibrium folding intermediate(s) along the folding pathway is less understood. In our present study, FTIR spectroscopy, far- and near-UV circular dichroism (CD), ANS and tryptophan fluorescence, near IR absorbance spectroscopy and dynamic light scattering (DLS) were used to study the structural and thermodynamic characteristics of the native (N), denatured (D) and intermediate state (X) of goat cytochorme c (cyt-c) induced by weak salt denaturants (LiBr, LiCl and LiClO4) at pH 6.0 and 25°C. The LiBr-induced denaturation of cyt-c measured by Soret absorption (Δε
400) and CD ([θ]409), is a three-step process, N ↔ X ↔ D. It is observed that the X state obtained along the denaturation pathway of cyt-c possesses common structural and thermodynamic characteristics of the molten globule (MG) state. The MG state of cyt-c induced by LiBr is compared for its structural and thermodynamic parameters with those found in other solvent conditions such as LiCl, LiClO4 and acidic pH. Our observations suggest: (1) that the LiBr-induced MG state of cyt-c retains the native Met80-Fe(III) axial bond and Trp59-propionate interactions; (2) that LiBr-induced MG state of cyt-c is more compact retaining the hydrophobic interactions in comparison to the MG states induced by LiCl, LiClO4 and 0.5 M NaCl at pH 2.0; and (3) that there exists heterogeneity of equilibrium intermediates along the unfolding pathway of cyt-c as highly ordered (X1), classical (X2) and disordered (X3), i.e., D ↔ X3 ↔ X2 ↔ X1 ↔ N. 相似文献
9.
《朊病毒》2013,7(4):236-242
Prion protein (PrP), the causative agent of Transmissible Spongiform Encephalopathies, is synthesized in the endoplasmic reticulum (ER) where it undergoes numerous covalent modifications. Here we investigate the interdependence and regulation of PrP oxidative folding, N-glycosylation, and GPI addition in diverse ER conditions. Our results show that formation of the single disulphide bond is a pivotal event, essential for PrP transport, and can occur post-translationally. Retarding its formation enhances N-glycosylation and GPI-anchoring. In contrast, lowering ER Ca2+ concentration inhibits N-glycosylation and GPI-anchoring. These data reveal tight interplays between the different ER covalent modifications, which collectively increase of PrP conformational diversity and may be important for its propagation. 相似文献
10.
Prion protein (PrP), the causative agent of transmissible spongiform encephalopathies, is synthesized in the endoplasmic reticulum (ER) where it undergoes numerous covalent modifications. Here we investigate the interdependence and regulation of PrP oxidative folding, N-glycosylation and GPI addition in diverse ER conditions. Our results show that formation of the single disulphide bond is a pivotal event, essential for PrP transport, and can occur post-translationally. Retarding its formation enhances N-glycosylation and GPI-anchoring. In contrast, lowering ER Ca2+ concentration inhibits N-glycosylation and GPI-anchoring. These data reveal tight interplays between the different ER covalent modifications, which collectively increase of PrP conformational diversity and may be important for its propagation.Key Words: Ca2+ homeostasis, ER-golgi transport, GPI-anchoring, N-glycosylation, oxidative folding, redox regulation 相似文献
11.
12.
The TTSS encoding ??translocator operon?? of Pseudomonas aeruginosa consists of a major translocator protein PopB, minor translocator protein PopD and their cognate chaperone PcrH. Far-UV CD spectra and secondary structure prediction servers predict an ??-helical model for PopB, PcrH and PopB?CPcrH complex. PopB itself forms a single species of higher order oligomer (15 mer) as seen from AUC, but in complex with PcrH, both monomeric (1:1) and oligomeric form exist. PopB has large solvent-exposed hydrophobic patches and exists as an unordered molten globule in its native state, but on forming complex with PcrH it gets transformed into an ordered molten globule. Tryptophan fluorescence spectrum indicates that PopB interacts with the first TPR region of dimeric PcrH to form a stable PopB?CPcrH complex that has a partial rigid structure with a large hydrodynamic radius and few tertiary contacts. The pH-dependent studies of PopB, PcrH and complex by ANS fluorescence, urea induced unfolding and thermal denaturation experiments prove that PcrH not only provides structural support to the ordered molten globule PopB in complex but also undergoes conformational change to assist PopB to pass through the needle complex of TTSS and form pores in the host cell membrane. ITC experiments show a strong affinity (Kd?~?0.37???M) of PopB for PcrH at pH 7.8, which reduces to ~0.68???M at pH 5.8. PcrH also loses its rigid tertiary structure at pH 5 and attains a molten globule conformation. This indicates that the decrease in pH releases PopB molecules and thus triggers the TTSS activation mechanism for the formation of a functional translocon. 相似文献
13.
Afshin Iram Tauqeer Alam Javed M. Khan Taqi A. Khan Rizwan H. Khan Aabgeena Naeem 《PloS one》2013,8(8)
Conformational alterations of bovine hemoglobin (Hb) upon sequential addition of glyoxal over a range of 0–90% v/v were investigated. At 20% v/v glyoxal, molten globule (MG) state of Hb was observed by altered tryptophan fluorescence, high ANS binding, existence of intact heme, native-like secondary structure as depicted by far-UV circular dichroism (CD) and ATR-FTIR spectra as well as loss in tertiary structure as confirmed by near-UV CD spectra. In addition, size exclusion chromatography analysis depicted that MG state at 20% v/v glyoxal corresponded to expanded pre-dissociated dimers. Aggregates of Hb were detected at 70% v/v glyoxal. These aggregates of Hb had altered tryptophan environment, low ANS binding, exposed heme, increased β-sheet secondary structure, loss in tertiary structure, enhanced thioflavin T (ThT) fluorescence and red shifted Congo Red (CR) absorbance. On incubating Hb with 30% v/v glyoxal for 0–20 days, advanced glycation end products (AGEs) were detected on day 20. These AGEs were characterised by enhanced tryptophan fluorescence at 450 nm, exposure of heme, increase in intermolecular β-sheets, enhanced ThT fluorescence and red shift in CR absorbance. Comet assay revealed aggregates and AGEs to be genotoxic in nature. Scanning electron microscopy confirmed the amorphous structure of aggregates and branched fibrils of AGEs. The transformation of α-helix to β-sheet usually alters the normal protein to amyloidogenic resulting in a variety of protein conformational disorders such as diabetes, prion and Huntington''s. 相似文献
14.
15.
Atiyatul Qadeer Gulam Rabbani Nida Zaidi Ejaz Ahmad Javed M. Khan Rizwan H. Khan 《PloS one》2012,7(11)
The molten globule (MG) state of proteins is widely detected through binding with 1-anilino-8-naphthalene sulphonate (ANS), a fluorescent dye. This strategy is based upon the assumption that when in molten globule state, the exposed hydrophobic clusters of protein are readily bound by the nonpolar anilino-naphthalene moiety of ANS molecules which then produce brilliant fluorescence. In this work, we explored the acid-induced unfolding pathway of chymopapain, a cysteine proteases from Carica papaya, by monitoring the conformational changes over a pH range 1.0–7.4 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). The spectroscopic measurements showed that although maximum ANS fluorescence intensity was observed at pH 1.0, however protein exhibited ∼80% loss of secondary structure which does not comply with the characteristics of a typical MG-state. In contrast at pH 1.5, chymopapain retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii and nearly 30-fold increase in ANS fluorescence with respect to the native state, indicating that MG-state exists at pH 1.5 and not at pH 1.0. ITC measurements revealed that ANS molecules bound to chymopapain via hydrophobic interaction were more at pH 1.5 than at pH 1.0. However, a large number of ANS molecules were also involved in electrostatic interaction with protein at pH 1.0 which, together with hydrophobically interacted molecules, may be responsible for maximum ANS fluorescence. We conclude that maximum ANS-fluorescence alone may not be the criteria for determining the MG of chymopapain. Hence a comprehensive structural analysis of the intermediate is essentially required. 相似文献
16.
17.
Matthew T. Honaker Mauro Acchione Wei Zhang Bengt Mannervik William M. Atkins 《The Journal of biological chemistry》2013,288(25):18599-18611
The role of conformational ensembles in enzymatic reactions remains unclear. Discussion concerning “induced fit” versus “conformational selection” has, however, ignored detoxication enzymes, which exhibit catalytic promiscuity. These enzymes dominate drug metabolism and determine drug-drug interactions. The detoxication enzyme glutathione transferase A1–1 (GSTA1–1), exploits a molten globule-like active site to achieve remarkable catalytic promiscuity wherein the substrate-free conformational ensemble is broad with barrierless transitions between states. A quantitative index of catalytic promiscuity is used to compare engineered variants of GSTA1–1 and the catalytic promiscuity correlates strongly with characteristics of the thermodynamic partition function, for the substrate-free enzymes. Access to chemically disparate transition states is encoded by the substrate-free conformational ensemble. Pre-steady state catalytic data confirm an extension of the conformational selection model, wherein different substrates select different starting conformations. The kinetic liability of the conformational breadth is minimized by a smooth landscape. We propose that “local” molten globule behavior optimizes detoxication enzymes. 相似文献
18.
19.
A mixture of 4-chloro-1-butanol and 2,2,2-Trifluoroethanol (TFE) has been used to generate Molten globule (MG) state of structurally
homologous but functionally different proteins bovine α-lactalbumin and hen egg-white lysozyme. The thermal denaturation was
done using UV–Visible spectroscopy. From UV–Visible profile, thermal transition was not observed beyond a particular concentration.
There was an indication of molten globule state in case of α-lactalbumin from circular dichroism experiments. By intrinsic
tryptophan fluorescence, acrylamide and potassium iodide quenching, 8-anilino-naphthalene sulfonic acid (ANS) binding and
energy transfer studies the presence of molten globule state was confirmed. Quantitative characterization of MG state and
determining the binding thermodynamics of ANS to the MG state was done using Isothermal Titration Calorimetry (ITC). Results
show that α-lactalbumin exists in MG state at a particular concentration but lysozyme does not show features of MG state. 相似文献
20.
The high resolution dielectric spectra of semidilute solutions of apomyoglobin in native (N, pH = 5), acid-induced molten globule (A, pH = 4), and unfolded (UA, pH = 3) states have been measured in the range from 0.2 to 20 GHz. Based on a two-component mixture theory, we obtained the following hydration numbers per protein molecule: 590 ± 65 for N, 630 ± 73 for A, and 1110 ± 67 for UA. There was no clear difference between N and A states in contrast to the 25% reduction of helix content and the 50% reduction of heat capacity change upon unfolding. This suggests that the association of hydrophobic moieties might follow the disruption of secondary structures from N to A states. The measured hydration number of UA was close to that of the accessible water number (1340) of a protein molecule calculated for a fully extended structure, indicating that the structure of UA is extended but somewhat more compact than that of a fully extended state. 相似文献