protein complex
Dotaz
Zobrazit nápovědu
K udržování buněčné homeostázy je nutné, aby buněčné proteiny vytvářely složité a dynamické molekulární komplexy. Proto je i vysvětlení základních fyziologických procesů na molekulární úrovni založeno na studiu protein‑proteinových interakcí. Nejdříve probíhá kvalitativní analýza proteinových komplexů. Následně jsou identifikované proteinové interakce kvantifikovány po biochemické stránce. Detailní informace o strukturní podstatě daných protein‑proteinových interakcí pak mohou být získány pomocí krystalografických metod. Náhled do uspořádání proteinových komplexů na molekulární úrovni umožňuje racionálně navrhovat nové syntetické látky, které cíleně ovlivňují proteinové interakce a tím i nejrůznější fyziologické nebo patologické procesy. Tato souhrnná práce je zaměřena na popis nejčastěji používaných metod pro kvalitativní i kvantitativní hodnocení proteinových interakcí. Metody koimunoprecipitace (Co‑IP) a afinitní koprecipitace je možné využít jako prvotní nástroj pro identifikaci interakčních partnerů studovaného proteinu. Detailní biochemická analýza mezimolekulární interakce pak vyžaduje definování kinetických a termodynamických parametrů. Pro studium afinity dvou interakčních partnerů a kinetiky reakce je možné použít metodu rezonance povrchového plazmonu (surface plasmon resonance – SPR), pro studium afinity a inhibičního potenciálu inhibitorů metodu fluorescenční polarizace (FP) a pro detailní popis afinity a termodynamických parametrů interakce (∆G, ∆H a ∆S) metodu izotermální titrační kalorimetrie (isothermal titration calorimetry – ITC). Výzkum proteinových interakcí na molekulární úrovni je nejen významný pro základní výzkum, ale přináší i nové metodické přístupy, které otvírají další možnosti při racionálním navrhování nových terapeutických látek.
In order to maintain cellular homeostasis, cellular proteins coexist in complex and variable molecular assemblies. Therefore, understanding of major physiological processes at molecular level is based on analysis of protein‑protein interaction networks. Firstly, composition of the molecular assembly has to be qualitatively analyzed. In the next step, quantitative biochemical properties of the identified protein‑protein interactions are determined. Detailed information about the protein‑protein interaction interface can be obtained by crystallographic methods. Accordingly, the insight into the molecular architecture of these protein‑protein complexes allows us to rationally design new synthetic compounds that specifically influence various physiological or pathological processes by targeted modulation of protein interactions. This review is focused on description of the most used methods applied in both qualitative and quantitative analysis of protein‑protein interactions. Co‑immunoprecipitation and affinity co‑precipitation are basic methods designed for qualitative analysis of protein binding partners. Further biochemical analysis of the interaction requires definition of kinetic and thermodynamic parameters. Surface plasmon resonance (SPR) is used for description of affinity and kinetic profile of the interaction, fluorescence polarization (FP) method for fast determination of inhibition potential of inhibitors and isothermal titration calorimetry (ITC) for definition of thermodynamic parameters of the interaction (∆G, ∆H and ∆S). Besides the importance of uncovering the molecular basis of protein interactions for basic research, the same methodological approaches open new possibilities in rational design of novel therapeutic agents. Key words: protein interaction networks – co‑immunoprecipitation – pull‑down analysis – surface plasmon resonance – fluorescence polarization – isothermal titration calorimetry This work was supported by the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101) and by MH CZ – DRO (MMCI, 00209805). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers. Submitted: 31. 1. 2014 Accepted: 10. 3. 2014
- Klíčová slova
- koimunoprecipitace, izotermální titrační kalorimetrie, afinitní koprecipitace, pull-down analýza,
- MeSH
- fluorescenční polarizace metody MeSH
- imunoprecipitace metody MeSH
- kalorimetrie metody MeSH
- ligandy MeSH
- mapování interakce mezi proteiny * metody MeSH
- mapy interakcí proteinů MeSH
- povrchová plasmonová rezonance metody MeSH
- termodynamika MeSH
- vazba proteinů * MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
Robust photosynthesis in chloroplasts and cyanobacteria requires the participation of accessory proteins to facilitate the assembly and maintenance of the photosynthetic apparatus located within the thylakoid membranes. The highly conserved Ycf48 protein acts early in the biogenesis of the oxygen-evolving photosystem II (PSII) complex by binding to newly synthesized precursor D1 subunit and by promoting efficient association with the D2 protein to form a PSII reaction center (PSII RC) assembly intermediate. Ycf48 is also required for efficient replacement of damaged D1 during the repair of PSII. However, the structural features underpinning Ycf48 function remain unclear. Here we show that Ycf48 proteins encoded by the thermophilic cyanobacterium Thermosynechococcus elongatus and the red alga Cyanidioschyzon merolae form seven-bladed beta-propellers with the 19-aa insertion characteristic of eukaryotic Ycf48 located at the junction of blades 3 and 4. Knowledge of these structures has allowed us to identify a conserved "Arg patch" on the surface of Ycf48 that is important for binding of Ycf48 to PSII RCs but also to larger complexes, including trimeric photosystem I (PSI). Reduced accumulation of chlorophyll in the absence of Ycf48 and the association of Ycf48 with PSI provide evidence of a more wide-ranging role for Ycf48 in the biogenesis of the photosynthetic apparatus than previously thought. Copurification of Ycf48 with the cyanobacterial YidC protein insertase supports the involvement of Ycf48 during the cotranslational insertion of chlorophyll-binding apopolypeptides into the membrane.
Efficient assembly and repair of the oxygen-evolving photosystem II (PSII) complex is vital for maintaining photosynthetic activity in plants, algae, and cyanobacteria. How chlorophyll is delivered to PSII during assembly and how vulnerable assembly complexes are protected from photodamage are unknown. Here, we identify a chlorophyll and β-carotene binding protein complex in the cyanobacterium Synechocystis PCC 6803 important for formation of the D1/D2 reaction center assembly complex. It is composed of putative short-chain dehydrogenase/reductase Ycf39, encoded by the slr0399 gene, and two members of the high-light-inducible protein (Hlip) family, HliC and HliD, which are small membrane proteins related to the light-harvesting chlorophyll binding complexes found in plants. Perturbed chlorophyll recycling in a Ycf39-null mutant and copurification of chlorophyll synthase and unassembled D1 with the Ycf39-Hlip complex indicate a role in the delivery of chlorophyll to newly synthesized D1. Sequence similarities suggest the presence of a related complex in chloroplasts.
Light quality significantly influences plant metabolism, growth and development. Recently, we have demonstrated that leaves of barley and other plant species grown under monochromatic green light (500-590 nm) accumulated a large pool of chlorophyll a (Chl a) intermediates with incomplete hydrogenation of their phytyl chains. In this work, we studied accumulation of these geranylgeranylated Chls a and b in pigment-protein complexes (PPCs) of Arabidopsis plants acclimated to green light and their structural-functional consequences on the photosynthetic apparatus. We found that geranylgeranylated Chls are present in all major PPCs, although their presence was more pronounced in light-harvesting complex II (LHCII) and less prominent in supercomplexes of photosystem II (PSII). Accumulation of geranylgeranylated Chls hampered the formation of PSII and PSI super- and megacomplexes in the thylakoid membranes as well as their assembly into chiral macrodomains; it also lowered the temperature stability of the PPCs, especially that of LHCII trimers, which led to their monomerization and an anomaly in the photoprotective mechanism of non-photochemical quenching. Role of geranylgeranylated Chls in adverse effects on photosynthetic apparatus of plants acclimated to green light is discussed.
... PROTEIN CD69 11 -- 1.3.1. Struktura proteinu CD69 11 -- I.3.2. ... ... HSP - PROTEINY TEPELNÉHO ŠOKU 16 -- 1.4.1. ... ... HSP60 rodina proteinů tepelného šoku - chaperoniny 16 -- I.5. ... ... Its Association with a GTP Binding Protein and Biochemical -- Requirements for Its Expression. [18] Sancho ...
57 listů : ilustrace ; 30 cm
Diplomová práce, která se zaměřila na možnou vazbu mezi Hsp65 a z něj odvozených peptidů na protein CD69 a na úlohu chaperoninu Hsp60.
- MeSH
- bakteriální proteiny MeSH
- chaperoniny MeSH
- chemické techniky analytické MeSH
- leukocyty MeSH
- ligandy MeSH
- proteiny teplotního šoku MeSH
- vazba proteinů MeSH
- Publikační typ
- vysokoškolské kvalifikační práce MeSH
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- biochemie
- alergologie a imunologie
This study combines mRNA and protein analysis using cDNA and antibody microarray techniques, respectively. These create a novel, integrated perspective into cellular molecular profiles. The aims of this study were to establish a reliable way of integrating these two approaches in order to obtain complex molecular profiles of the cell and to find suitable methods to normalize the data obtained using these approaches.
Antibody microarray and cDNA microarray techniques were used to study expression alterations in HL-60 cells that were differentiated into granulocytes using all-trans retinoic acid (ATRA). We selected this model to evaluate this combined profiling technique because the expression levels of most of the mRNA and protein species in these cells are not altered; therefore it is easier to track and define those species that are changed. The proteins whose levels were altered included c-myc, c-jun, Pyk2, FAK, PKC, TRF1, NF-kappaB and certain caspase types. These proteins are involved in apoptosis and hematopoietic differentiation pathways, and some have also been reported to have oncogenic potential. We compared the results obtained using the two methods, verified them by immunoblotting analysis, and devised normalization approaches.
This is one of the first demonstrations that a combination of antibody microarray and cDNA microarray techniques is required for complex molecular profiling of cells based on multiple parameters. This approach allows a more detailed molecular phenotype of the given sample to be obtained. The results obtained using a combination of the two profiling methods are consistent with those from previous studies that used more traditional methods.
Keywords: microarray, cell profiling, protein expression, mRNA expression, HL-60.- MeSH
- čipová analýza proteinů MeSH
- financování organizované MeSH
- fokální adhezní kinasa 2 analýza MeSH
- geny myc MeSH
- HL-60 buňky MeSH
- lidé MeSH
- messenger RNA analýza MeSH
- protein TRF1 analýza MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- tretinoin farmakologie MeSH
- Check Tag
- lidé MeSH
... Motifs of Protein Structure -- Few general principles emerged from the first protein structure -- The ... ... Membrane Proteins 201 -- Membrane proteins are difficult to crystallize 202 -- Bacteriorhodopsin contains ... ... mutations in the GTP-binding loops 226 -- The molecular basis of autophosphorylation of viral p21 226 -- Protein-protein ... ... Prediction, Engineering, and Design of -- Protein Structures 247 -- Prediction of protein structure from ... ... of protein molecules 269 -- Protein crystals are difficult to grow 270 -- X-ray sources are either monochromatic ...
xv, 302 stran : ilustrace ; 28 cm
