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1.
Food Chem ; 402: 134250, 2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36126583

RESUMO

Magnetic nanoparticles (MNPs) have a dual role in acting as magnetic and sonosensitizer agents, which can combine the synergistic effects of microwave and ultrasonic waves. To study the effects of MNPs combined ultrasonic-microwave thawing (NUMT) on the water holding capacity (WHC), oxidation of protein and lipid, and protein conformation, jumbo squid mantles were subjected to cold storage thawing (CST), MNPs combined ultrasonic thawing (NUT), MNPs combined microwave thawing (NMT) and NUMT. Results showed that NUMT treatment had a higher WHC, lower oxidation, effectively reduced myofibrillar protein aggregation and degradation, and stabilized the structure of the protein of the jumbo squid. The muscle fiber structure of NUMT treated jumbo squid mantles was dense, orderly with a smooth surface, and the fiber network gaps were small and uniformly distributed. This study shows that NUMT can ameliorate the thawing qualities of jumbo squid, and is an effectively thawing method.


Assuntos
Nanopartículas , Ultrassom , Animais , Ultrassom/métodos , Água/química , Micro-Ondas , Agregados Proteicos , Decapodiformes/química , Proteínas , Conformação Proteica , Fibras Musculares Esqueléticas , Lipídeos
2.
Food Chem ; 402: 134230, 2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36130431

RESUMO

Tracking the dynamic changes in the structure of kidney bean protein isolate (KPI) during extreme pH-shifting can reveal the different mechanisms that drive the unfolding and refolding of the protein from a conformational perspective and elucidate the relationship between its structure and function. The secondary and tertiary structures of KPI were analyzed using multispectral techniques. The results showed that acidic-shifting affected the hydrophobic interactions of KPI molecules, whereas alkaline-shifting affected hydrogen bonding and electrostatic interactions of the molecules. Therefore, alkaline-shifting was more likely to affect KPI conformation. SEM revealed that pH-shifting transformed the sheet structure of KPI into spheres and rods; moreover, it improved the surface hydrophobicity, thermal stability, emulsification, foaming, and antioxidant properties of KPI. In summary, each pH-shifting stage disrupts a different intermolecular force, resulting in protein conformational diversity, while structural changes further affect function. Therefore, pH-shifting treatment broadens the applications scope of KPI in the food industry.


Assuntos
Phaseolus , Phaseolus/genética , Phaseolus/química , Antioxidantes , Concentração de Íons de Hidrogênio , Conformação Proteica , Interações Hidrofóbicas e Hidrofílicas , Dobramento de Proteína
3.
Methods Mol Biol ; 2552: 61-79, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346585

RESUMO

The immune systems protect vertebrates from foreign molecules or antigens, and antibodies are important mediators of this system. The sequences and structural features of antibodies vary depending on species. Many of antibodies from vertebrates, including camelids, have both heavy and light chain variable domains, but camelids also have antibodies that lack the light chains. In antibodies that lack light chains, the C-terminal variable region is called the VHH domain. Antibodies recognize antigens through six complementarity-determining regions (CDRs). The third CDR of the heavy chain (CDR-H3) is at the center of the antigen-binding site and is diverse in terms of sequence and structure. Due to the importance of antibodies in basic science as well as in medical applications, there have been many studies of CDR-H3s of antibodies that possess both light and heavy chains. However, nature of CDR-H3s of single-domain VHH antibodies is less well studied. In this chapter, we describe current knowledge of sequence-structure-function correlations of single-domain VHH antibodies with emphasis on CDR-H3. Based on the 370 crystal structures in the Protein Data Bank, we also attempt structural classification of CDR-H3 in single-domain VHH antibodies and discuss lessons learned from the ever-increasing number of the structures.


Assuntos
Anticorpos de Domínio Único , Animais , Anticorpos de Domínio Único/química , Modelos Moleculares , Regiões Determinantes de Complementaridade , Anticorpos/química , Bases de Dados de Proteínas , Antígenos , Conformação Proteica
4.
Methods Mol Biol ; 2552: 109-124, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346588

RESUMO

Complex and coordinated dynamics are closely connected with protein functions, including the binding of antibodies to antigens. Knowledge of such dynamics could improve the design of antibodies. Molecular dynamics (MD) simulations provide a "computational microscope" that can resolve atomic motions and inform antibody design efforts.


Assuntos
Anticorpos , Simulação de Dinâmica Molecular , Anticorpos/química , Antígenos , Proteínas/química , Conformação Proteica
5.
Methods Mol Biol ; 2552: 125-139, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346589

RESUMO

This chapter describes the application of constrained geometric simulations for prediction of antibody structural dynamics. We utilize constrained geometric simulations method FRODAN, which is a low computational complexity alternative to molecular dynamics (MD) simulations that can rapidly explore flexible motions in protein structures. FRODAN is highly suited for conformational dynamics analysis of large proteins, complexes, intrinsically disordered proteins, and dynamics that occurs on longer biologically relevant time scales that are normally inaccessible to classical MD simulations. This approach predicts protein dynamics at an all-atom scale while retaining realistic covalent bonding, maintaining dihedral angles in energetically good conformations while avoiding steric clashes in addition to performing other geometric and stereochemical criteria checks. In this chapter, we apply FRODAN to showcase its applicability for probing functionally relevant dynamics of IgG2a, including large-amplitude domain-domain motions and motions of complementarity determining region (CDR) loops. As was suggested in previous experimental studies, our simulations show that antibodies can explore a large range of conformational space.


Assuntos
Proteínas Intrinsicamente Desordenadas , Simulação de Dinâmica Molecular , Conformação Proteica , Regiões Determinantes de Complementaridade , Anticorpos
6.
Methods Mol Biol ; 2568: 75-101, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36227563

RESUMO

Recognition of the growing importance of RNA as a target for therapeutic or diagnostic ligands brings the importance of computational predictions of docking poses to such receptors to the forefront. Most docking programs have been optimized for protein targets, based on a relatively rich pool of known docked protein structures. Unfortunately, despite progress, numbers of known docked RNA complexes are low and the accuracy of the computational predictions trained on those inadequate samples lags behind that achieved for proteins. Compared to proteins, RNA structures generally have fewer docking pockets, have less diverse electrostatic surfaces, and are more flexible, raising the possibility of producing only transiently available good docking targets. We are presenting a docking prediction protocol that adds molecular dynamics simulations before and after the actual docking in order to explore the conformational space of the target RNA and then to reevaluate the stability of the predicted RNA-ligand complex. In this way we are attempting to overcome important limitations of the docking programs: the rigid (fully or mostly) target structure and imperfect nature of the docking scoring functions.


Assuntos
Simulação de Dinâmica Molecular , Proteínas , Sítios de Ligação , Ligantes , Conformação Molecular , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Proteínas/química , RNA/metabolismo
7.
Methods Mol Biol ; 2568: 193-211, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36227570

RESUMO

RNA three-dimensional structures provide rich and vital information for understanding their functions. Recent advances in cryogenic electron microscopy (cryo-EM) allow structure determination of RNAs and ribonucleoprotein (RNP) complexes. However, limited global and local resolutions of RNA cryo-EM maps pose great challenges in tracing RNA coordinates. The Rosetta-based "auto-DRRAFTER" method builds RNA models into moderate-resolution RNA cryo-EM density as part of the Ribosolve pipeline. Here, we describe a step-by-step protocol for auto-DRRAFTER using a glycine riboswitch from Fusobacterium nucleatum as an example. Successful implementation of this protocol allows automated RNA modeling into RNA cryo-EM density, accelerating our understanding of RNA structure-function relationships. Input and output files are being made available at https://github.com/auto-DRRAFTER/springer-chapter .


Assuntos
RNA , Riboswitch , Microscopia Crioeletrônica/métodos , Glicina , Modelos Moleculares , Conformação Proteica , Ribonucleoproteínas
8.
Acta Crystallogr D Struct Biol ; 78(Pt 11): 1303-1314, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36322415

RESUMO

AlphaFold has recently become an important tool in providing models for experimental structure determination by X-ray crystallography and cryo-EM. Large parts of the predicted models typically approach the accuracy of experimentally determined structures, although there are frequently local errors and errors in the relative orientations of domains. Importantly, residues in the model of a protein predicted by AlphaFold are tagged with a predicted local distance difference test score, informing users about which regions of the structure are predicted with less confidence. AlphaFold also produces a predicted aligned error matrix indicating its confidence in the relative positions of each pair of residues in the predicted model. The phenix.process_predicted_model tool downweights or removes low-confidence residues and can break a model into confidently predicted domains in preparation for molecular replacement or cryo-EM docking. These confidence metrics are further used in ISOLDE to weight torsion and atom-atom distance restraints, allowing the complete AlphaFold model to be interactively rearranged to match the docked fragments and reducing the need for the rebuilding of connecting regions.


Assuntos
Software , Modelos Moleculares , Cristalografia por Raios X , Conformação Proteica , Microscopia Crioeletrônica
9.
Molecules ; 27(21)2022 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-36364032

RESUMO

Human transthyretin (hTTR) can form amyloid deposits that accumulate in nerves and organs, disrupting cellular function. Molecules such as tafamidis that bind to and stabilize the TTR tetramer can reduce such amyloid formation. Here, we studied the interaction of VCP-6 (2-((3,5-dichlorophenyl)amino)benzoic acid) with hTTR. VCP-6 binds to hTTR with 5 times the affinity of the cognate ligand, thyroxine (T4). The structure of the hTTR:VCP-6 complex was determined by X-ray crystallography at 1.52 Šresolution. VCP-6 binds deeper in the binding channel than T4 with the 3',5'-dichlorophenyl ring binding in the 'forward' mode towards the channel centre. The dichlorophenyl ring lies along the 2-fold axis coincident with the channel centre, while the 2-carboxylatephenylamine ring of VCP-6 is symmetrically displaced from the 2-fold axis, allowing the 2-carboxylate group to form a tight intermolecular hydrogen bond with Nζ of Lys15 and an intramolecular hydrogen bond with the amine of VCP-6, stabilizing its conformation and explaining the greater affinity of VCP-6 compared to T4. This arrangement maintains optimal halogen bonding interactions in the binding sites, via chlorine atoms rather than iodine of the thyroid hormone, thereby explaining why the dichloro substitution pattern is a stronger binder than either the diiodo or dibromo analogues.


Assuntos
Amiloidose , Pré-Albumina , Humanos , Pré-Albumina/metabolismo , Conformação Proteica , Modelos Moleculares , Sítios de Ligação , Cristalografia por Raios X , Amiloide , Proteínas Amiloidogênicas , Halogênios
10.
Protein Sci ; 31(9): e4403, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36367084

RESUMO

Calgranulin C performs antimicrobial activity in the human immune response by sequestering Zn(II). This biological function is afforded with the aid of two structurally distinct Ca(II)-binding EF hand motifs, wherein one of which bears an unusual amino acid sequence. Here, we utilize solution state NMR relaxation measurements to investigate the mechanism of Ca(II)-modulated enhancement of Zn(II) sequestration by calgranulin C. Using C13 /N15 CPMG dispersion experiments we have measured pH-dependent major and minor state populations exchanging on micro-to-millisecond timescale. This conformational exchange takes place exclusively in the Ca(II)-bound state and can be mapped to residues located in the EF-I loop and the linker between the tandem EF hands. Molecular dynamics (MD) simulations spanning nano-to-microsecond timescale offer insights into the role of pH-dependent electrostatic interactions in EF-hand dynamics. Our results suggest a pH-regulated dynamic equilibrium of conformations that explore a range of "closed" and partially "open" sidechain configurations within the Zn(II) binding site. We propose a novel mechanism by which Ca(II) binding to a non-canonical EF loop regulates its flexibility and tunes the antimicrobial activity of calgranulin C.


Assuntos
Anti-Infecciosos , Motivos EF Hand , Humanos , Conformação Proteica , Modelos Moleculares , Complexo Antígeno L1 Leucocitário/metabolismo , Zinco/metabolismo , Cálcio/metabolismo
11.
Sci Rep ; 12(1): 19057, 2022 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-36352011

RESUMO

Intrinsically disordered proteins (IDP) are at the center of numerous biological processes, and attract consequently extreme interest in structural biology. Numerous approaches have been developed for generating sets of IDP conformations verifying a given set of experimental measurements. We propose here to perform a systematic enumeration of protein conformations, carried out using the TAiBP approach based on distance geometry. This enumeration was performed on two proteins, Sic1 and pSic1, corresponding to unphosphorylated and phosphorylated states of an IDP. The relative populations of the obtained conformations were then obtained by fitting SAXS curves as well as Ramachandran probability maps, the original finite mixture approach RamaMix being developed for this second task. The similarity between profiles of local gyration radii provides to a certain extent a converged view of the Sic1 and pSic1 conformational space. Profiles and populations are thus proposed for describing IDP conformations. Different variations of the resulting gyration radius between phosphorylated and unphosphorylated states are observed, depending on the set of enumerated conformations as well as on the methods used for obtaining the populations.


Assuntos
Inosina Difosfato , Proteínas Intrinsicamente Desordenadas , Proteínas Intrinsicamente Desordenadas/química , Espalhamento a Baixo Ângulo , Difração de Raios X , Conformação Proteica
12.
Nat Struct Mol Biol ; 29(11): 1056-1067, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36344848

RESUMO

Most proteins fold into 3D structures that determine how they function and orchestrate the biological processes of the cell. Recent developments in computational methods for protein structure predictions have reached the accuracy of experimentally determined models. Although this has been independently verified, the implementation of these methods across structural-biology applications remains to be tested. Here, we evaluate the use of AlphaFold2 (AF2) predictions in the study of characteristic structural elements; the impact of missense variants; function and ligand binding site predictions; modeling of interactions; and modeling of experimental structural data. For 11 proteomes, an average of 25% additional residues can be confidently modeled when compared with homology modeling, identifying structural features rarely seen in the Protein Data Bank. AF2-based predictions of protein disorder and complexes surpass dedicated tools, and AF2 models can be used across diverse applications equally well compared with experimentally determined structures, when the confidence metrics are critically considered. In summary, we find that these advances are likely to have a transformative impact in structural biology and broader life-science research.


Assuntos
Biologia Computacional , Furilfuramida , Biologia Computacional/métodos , Sítios de Ligação , Proteínas/química , Bases de Dados de Proteínas , Conformação Proteica
13.
Biomolecules ; 12(11)2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36358915

RESUMO

Intrinsically disordered proteins (IDPs) lack well-defined 3D structures and can only be described as ensembles of different conformations. This high degree of flexibility allows them to interact promiscuously and makes them capable of fulfilling unique and versatile regulatory roles in cellular processes. These functional benefits make IDPs widespread in nature, existing in every living organism from bacteria and fungi to plants and animals. Due to their open and exposed structural state, IDPs are much more prone to proteolytic degradation than their globular counterparts. Therefore, the purification of recombinant IDPs requires extra care and caution, such as maintaining low temperature throughout the purification, the use of protease inhibitor cocktails and fast workflow. Even so, in the case of long IDP targets, the appearance of truncated by-products often seems unavoidable. The separation of these unwanted proteins can be very challenging due to their similarity to the parent target protein. Here, we describe a tandem-tag purification method that offers a remedy to this problem. It contains only common affinity-chromatography steps (HisTrap and Heparin) to ensure low cost, easy access and scaling-up for possible industrial use. The effectiveness of the method is demonstrated with four examples, Tau-441 and two of its fragments and the transactivation domain (AF1) of androgen receptor.


Assuntos
Proteínas Intrinsicamente Desordenadas , Cromatografia de Afinidade , Proteínas Intrinsicamente Desordenadas/química , Conformação Proteica
14.
BMC Bioinformatics ; 23(1): 456, 2022 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-36324073

RESUMO

BACKGROUND: Ligand-protein interactions play a key role in defining protein function, and detecting natural ligands for a given protein is thus a very important bioengineering task. In particular, with the rapid development of AI-based structure prediction algorithms, batch structural models with high reliability and accuracy can be obtained at low cost, giving rise to the urgent requirement for the prediction of natural ligands based on protein structures. In recent years, although several structure-based methods have been developed to predict ligand-binding pockets and ligand-binding sites, accurate and rapid methods are still lacking, especially for the prediction of ligand-binding regions and the spatial extension of ligands in the pockets. RESULTS: In this paper, we proposed a multilayer dynamics perturbation analysis (MDPA) method for predicting ligand-binding regions based solely on protein structure, which is an extended version of our previously developed fast dynamic perturbation analysis (FDPA) method. In MDPA/FDPA, ligand binding tends to occur in regions that cause large changes in protein conformational dynamics. MDPA, examined using a standard validation dataset of ligand-protein complexes, yielded an averaged ligand-binding site prediction Matthews coefficient of 0.40, with a prediction precision of at least 50% for 71% of the cases. In particular, for 80% of the cases, the predicted ligand-binding region overlaps the natural ligand by at least 50%. The method was also compared with other state-of-the-art structure-based methods. CONCLUSIONS: MDPA is a structure-based method to detect ligand-binding regions on protein surface. Our calculations suggested that a range of spaces inside the protein pockets has subtle interactions with the protein, which can significantly impact on the overall dynamics of the protein. This work provides a valuable tool as a starting point upon which further docking and analysis methods can be used for natural ligand detection in protein functional annotation. The source code of MDPA method is freely available at: https://github.com/mingdengming/mdpa .


Assuntos
Algoritmos , Proteínas , Ligantes , Ligação Proteica , Reprodutibilidade dos Testes , Sítios de Ligação , Conformação Proteica , Proteínas/química
15.
J Gen Physiol ; 154(12)2022 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-36326620

RESUMO

A critical part of ion channel function is the ability to open and close in response to stimuli and thus conduct ions in a regulated fashion. While x-ray diffraction studies of ion channels suggested a general steric gating mechanism located at the helix bundle crossing (HBC), recent functional studies on several channels indicate that the helix bundle crossing is wide-open even in functionally nonconductive channels. Two NaK channel variants were crystallized in very different open and closed conformations, which served as important models of the HBC gating hypothesis. However, neither of these NaK variants is conductive in liposomes unless phenylalanine 92 is mutated to alanine (F92A). Here, we use NMR to probe distances at near-atomic resolution of the two NaK variants in lipid bicelles. We demonstrate that in contrast to the crystal structures, both NaK variants are in a fully open conformation, akin to Ca2+-bound MthK channel structure where the HBC is widely open. While we were not able to determine what a conductive NaK structure is like, our further inquiry into the gating mechanism suggests that the selectivity filter and pore helix are coupled to the M2 helix below and undergo changes in the structure when F92 is mutated. Overall, our data show that NaK exhibits coupling between the selectivity filter and HBC, similar to K+ channels, and has a more complex gating mechanism than previously thought, where the full opening of HBC does not lead to channel activation.


Assuntos
Ativação do Canal Iônico , Canais Iônicos , Ativação do Canal Iônico/fisiologia , Canais Iônicos/química , Íons , Conformação Proteica
16.
Nat Struct Mol Biol ; 29(11): 1068-1079, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36329286

RESUMO

ClpAP, a two-ring AAA+ protease, degrades N-end-rule proteins bound by the ClpS adaptor. Here we present high-resolution cryo-EM structures of Escherichia coli ClpAPS complexes, showing how ClpA pore loops interact with the ClpS N-terminal extension (NTE), which is normally intrinsically disordered. In two classes, the NTE is bound by a spiral of pore-1 and pore-2 loops in a manner similar to substrate-polypeptide binding by many AAA+ unfoldases. Kinetic studies reveal that pore-2 loops of the ClpA D1 ring catalyze the protein remodeling required for substrate delivery by ClpS. In a third class, D2 pore-1 loops are rotated, tucked away from the channel and do not bind the NTE, demonstrating asymmetry in engagement by the D1 and D2 rings. These studies show additional structures and functions for key AAA+ elements. Pore-loop tucking may be used broadly by AAA+ unfoldases, for example, during enzyme pausing/unloading.


Assuntos
Proteínas de Transporte , Endopeptidase Clp , Proteínas de Escherichia coli , ATPases Associadas a Diversas Atividades Celulares/química , Proteínas de Transporte/química , Endopeptidase Clp/química , Escherichia coli/química , Proteínas de Escherichia coli/química , Cinética , Chaperonas Moleculares/química , Conformação Proteica
17.
Commun Biol ; 5(1): 1249, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36376429

RESUMO

Isoflavonoids play important roles in plant defense and also exhibit a range of mammalian health-promoting activities. Their biosynthesis is initiated by two enzymes with unusual catalytic activities; 2-hydroxyisoflavanone synthase (2-HIS), a membrane-bound cytochrome P450 catalyzing a coupled aryl-ring migration and hydroxylation, and 2-hydroxyisoflavanone dehydratase (2-HID), a member of a large carboxylesterase family that paradoxically catalyzes dehydration of 2-hydroxyisoflavanones to isoflavone. Here we report the crystal structures of 2-HIS from Medicago truncatula and 2-HID from Pueraria lobata. The 2-HIS structure reveals a unique cytochrome P450 conformation and heme and substrate binding mode that facilitate the coupled aryl-ring migration and hydroxylation reactions. The 2-HID structure reveals the active site architecture and putative catalytic residues for the dual dehydratase and carboxylesterase activities. Mutagenesis studies revealed key residues involved in substrate binding and specificity. Understanding the structural basis of isoflavone biosynthesis will facilitate the engineering of new bioactive isoflavonoids.


Assuntos
Isoflavonas , Animais , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Conformação Proteica , Hidroliases/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Mamíferos
18.
PLoS Pathog ; 18(11): e1010947, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36342968

RESUMO

Prion strains in a given type of mammalian host are distinguished by differences in clinical presentation, neuropathological lesions, survival time, and characteristics of the infecting prion protein (PrP) assemblies. Near-atomic structures of prions from two host species with different PrP sequences have been determined but comparisons of distinct prion strains of the same amino acid sequence are needed to identify purely conformational determinants of prion strain characteristics. Here we report a 3.2 Å resolution cryogenic electron microscopy-based structure of the 22L prion strain purified from the brains of mice engineered to express only PrP lacking glycophosphatidylinositol anchors [anchorless (a) 22L]. Comparison of this near-atomic structure to our recently determined structure of the aRML strain propagated in the same inbred mouse reveals that these two mouse prion strains have distinct conformational templates for growth via incorporation of PrP molecules of the same sequence. Both a22L and aRML are assembled as stacks of PrP molecules forming parallel in-register intermolecular ß-sheets and intervening loops, with single monomers spanning the ordered fibril core. Each monomer shares an N-terminal steric zipper, three major arches, and an overall V-shape, but the details of these and other conformational features differ markedly. Thus, variations in shared conformational motifs within a parallel in-register ß-stack fibril architecture provide a structural basis for prion strain differentiation within a single host genotype.


Assuntos
Príons , Animais , Camundongos , Microscopia Crioeletrônica , Genótipo , Proteínas Priônicas/genética , Príons/metabolismo , Conformação Proteica
19.
Sci Rep ; 12(1): 19613, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36379962

RESUMO

Coelenterazine-v (CTZ-v), a synthetic vinylene-bridged π-extended derivative, is able to significantly alter bioluminescence spectra of different CTZ-dependent luciferases and photoproteins by shifting them towards longer wavelengths. However, Ca2+-regulated photoproteins activated with CTZ-v display very low bioluminescence activities that hampers its usage as a substrate of photoprotein bioluminescence. Here, we report the crystal structure of semi-synthetic Ca2+-discharged obelin-v bound with the reaction product determined at 2.1 Å resolution. Comparison of the crystal structure of Ca2+-discharged obelin-v with those of other obelins before and after bioluminescence reaction reveals no considerable changes in the overall structure. However, the drastic changes in CTZ-binding cavity are observed owing to the completely different reaction product, coelenteramine-v (CTM-v). Since CTM-v is certainly the main product of obelin-v bioluminescence and is considered to be a product of the "dark" pathway of dioxetanone intermediate decomposition, it explains the low bioluminescence activity of obelin and apparently of other photoproteins with CTZ-v.


Assuntos
Cálcio na Dieta , Cálcio , Cálcio/metabolismo , Conformação Proteica , Proteínas Luminescentes/metabolismo , Medições Luminescentes
20.
J Phys Chem B ; 126(45): 9176-9186, 2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36331868

RESUMO

Structural characterization of intrinsically disordered proteins (IDPs) requires a concerted effort between experiments and computations by accounting for their conformational heterogeneity. Given the diversity of experimental tools providing local and global structural information, constructing an experimental restraint-satisfying structural ensemble remains challenging. Here, we use the disordered N-terminal domain (NTD) of the estrogen receptor alpha (ERalpha) as a model system to combine existing small-angle X-ray scattering (SAXS) and hydroxyl radical protein footprinting (HRPF) data and newly acquired solvent accessibility data via D2O-induced fluorine chemical shifting (DFCS) measurements. A new set of DFCS data for the solvent exposure of a set of 12 amino acid positions were added to complement previously acquired HRPF measurements for the solvent exposure of the other 16 nonoverlapping amino acids, thereby improving the NTD ensemble characterization considerably. We also found that while choosing an initial ensemble of structures generated from a different atomic-level force field or sampling/modeling method can lead to distinct contact maps even when the same sets of experimental measurements were used for ensemble-fitting, comparative analyses from these initial ensembles reveal commonly recurring structural features in their ensemble-averaged contact map. Specifically, nonlocal or long-range transient interactions were found consistently between the N-terminal segments and the central region, sufficient to mediate the conformational ensemble and regulate how the NTD interacts with its coactivator proteins.


Assuntos
Receptor alfa de Estrogênio , Proteínas Intrinsicamente Desordenadas , Espalhamento a Baixo Ângulo , Difração de Raios X , Conformação Proteica , Flúor , Proteínas Intrinsicamente Desordenadas/química , Solventes
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