6 research outputs found
Engineering the thermostability of a TIM-barrel enzyme by rational family shuffling
a b s t r a c t A possible approach to generate enzymes with an engineered temperature optimum is to create chimeras of homologous enzymes with different temperature optima. We tested this approach using two family-10 xylanases from Thermotoga maritima: the thermophilic xylanase A catalytic domain (TmxAcat, T opt = 68°C), and the hyperthermophilic xylanase B (TmxB, T opt = 102°C). Twenty-one different chimeric constructs were created by mimicking family shuffling in a rational manner. The measured temperature optima of the 16 enzymatically active chimeras do not monotonically increase with the percentage of residues coming from TmxB. Only four chimeras had a higher temperature optimum than TmxAcat, the most stable variant (T opt = 80°C) being the one in which both terminal segments came from TmxB. Further analysis suggests that the interaction between the N-and C-terminal segments has a disproportionately high contribution to the overall thermostability. The results may be generalizable to other enzymes where the N-and C-termini are in contact. Ó 2008 Elsevier Inc. All rights reserved. Microorganisms occur in almost all environments on Earth, including high-temperature environments such as hot springs. In most cases, proteins from (hyper)thermophilic organisms have been found to be structurally similar to their mesophilic counterparts, except for minor differences The (b/a) 8 -barrel fold, which was found in triose-phosphate isomerase, and is therefore also known as the TIM-barrel fold, is the most common enzyme fold In the present study, we used two family-10 xylanases from the hyperthermophilic eubacterium Thermotoga maritima MSB8 with widely different temperature optima as starting points for rational family shuffling Materials and methods Construction of chimeric enzymes. Genes xynAcat (GenBank Accession No. Z46264, basepairs 1340-2323) and xynB (GenBank Accession No. AAD35164) encoding TmxB and TmxAcat were PCR 0006-291X/$ -see front matter
Összefüggés a fehérjék stabilitása, konformációs flexibilitása és működése között = Relationship between stability, conformational flexibility and function of proteins
HomolĂłg hĹ‘kedvelĹ‘, mezofil Ă©s hidegtűrĹ‘ enzimek sorozatainak szerkezeti, szervezĹ‘dĂ©si (folding) Ă©s funkcionális (reakciĂł kinetikai) összehasonlĂtĂł vizsgálatát vĂ©geztĂĽk el. A katalitikus reakciĂł enzimkinetikai paramĂ©tereinek a hĹ‘mĂ©rsĂ©klet Ă©s a konformáciĂłs fluktuáciĂłk (relaxáciĂłs spektrum) fĂĽggvĂ©nyĂ©ben törtĂ©nĹ‘ analĂzisĂ©vel megállapĂtottuk, hogy a gliceraldehid-3-foszfát dehidrogenáz enzim alegysĂ©gei közötti allosztĂ©rikus jeltovábbĂtásban a molekula dinamikus tulajdonságainak meghatározĂł szerepe van. A több domĂ©nbĹ‘l állĂł modellfehĂ©rjĂ©k (IPMDH, PGK) esetĂ©ben feltártuk a domĂ©nzárĂłdás atomi szintű lĂ©pĂ©seinek összefĂĽggĂ©sĂ©t a katalitikus folyamat egyes elemeivel. A fehĂ©rjĂ©k folding-refolding folyamatainak kinetikai analĂzĂsĂ©vel több, a hĹ‘stabilitás mechanizmusát Ă©rtelmezĹ‘ Ăşj megállapĂtást tettĂĽnk. Vizsgáltuk a Thermotoga maritima eubaktĂ©riumbĂłl származĂł, hasonlĂłságot mutatĂł termofil Ă©s hipertermofil xilanázok hĹ‘stabilitása közötti kĂĽlönbsĂ©g szerkezeti hátterĂ©t. IrányĂtott family shuffling segĂtsĂ©gĂ©vel számos kimĂ©ra enzimet állĂtottunk elĹ‘ a kĂ©t vad tĂpusĂş xilanázbĂłl. A termofil xilanáz enzim terminális rĂ©giĂłit lecserĂ©lve a hipertermofil xilanáz enzim terminális rĂ©giĂłira, a termofil enzim hĹ‘stabilitását 12 oC-kal megnöveltĂĽk. A lĂ©trehozott kimĂ©ra konstrukciĂłk arra világĂtottak rá, hogy a terminális rĂ©giĂłk közötti kölcsönhatás stabilizálásával megnövelhetĹ‘ azon fehĂ©rjĂ©k hĹ‘stabilitása, amelyek terminális rĂ©giĂłi megfelelĹ‘ közelsĂ©gben találhatĂłak. | Comparative structural, folding and functional studies were completed using sets of homologous thermophilic, mesophilic, and psychrotropic enzymes. It was revealed that the dynamic features of the enzyme molecule are essential for the mechanism of intramolecular, allosteric signal transduction between subunits of glyceraldehyde-3-phosphate dehydrogenase. The steps of domain closure, at the atomic level, were related to individual elements of the catalytic process in the case of multidomain enzymes (IPMDH, PGK). A novel mechanistic interpretation was developed based on kinetic analyses of folding-refolding processes. The structural background of the difference in heat stabilities between homologous thermophilic and hyperthermophilic xylanases from Thermotoga maritima was studied. Several chimeric constructs of the two parental enzymes were produced by directed family shuffling. A significant increase in heat stability was achieved in the case of one construct. Exchanging the terminal regions of the thermophilic enzyme by the respective fragments of the hyperthermophilic xylanase, the heat stability of the enzyme increased by 12 oC. Characterization of chimeric constructs showed that the interactions between terminal regions have an important role in stabilizing protein structure, and based on this observation, heat stability can be increased in proteins in which the terminal regions are in close proximity
Enzimreakciók vizsgálata a moduláris szerveződés, az atomi kölcsönhatás és a kvantummechanika szintjein. A fehérje biofizika tudományos iskolája = Insight into the Enzyme Action at Levels of modular Organization, Atomic Interactions and Quantum-Mechanics. School of Protein Biophysics
Az elmĂşlt 3 Ă©v koherens kutatĂł munkája során szĂĽlettek speciális tudományos eredmĂ©nyek Ă©s levontunk ezekbĹ‘l általános következtetĂ©seket is. Munkánk mĂ©rlege a nemzetközi folyĂłiratokban megjelent 30 közlemĂ©ny összesen 130 IF-al. Molekuláris immunolĂłgiai kutatásaink keretĂ©ben meghatároztuk 4 komplement proteáz Ă©s a C1-inhibitor szerkezetĂ©t, kĂĽlönösen az utĂłbbi hozott számunkra nagy nemzetközi elismerĂ©st. A szerkezetek Ă©s funkcionális eredmĂ©nyeink alapján általánosan elfogadott aktiválási modellt dolgoztunk ki a komplement rendszer lektin Ăştjának szabályozási mechanizmusára. JelentĹ‘snek tartjuk a C1-inhibitor heparin által törtĂ©nĹ‘ potencirozásának mechanizmusára javasolt, szerkezeti alapĂş modell kidolgozását, a flagellin fehĂ©rje egyik rendezetlen szakaszának export szignálkĂ©nt törtĂ©nĹ‘ azonosĂtását (szabadalom is szĂĽletett belĹ‘le), a foszfoglicerátkináz enzim domĂ©n zárĂłdásban rĂ©sztvevĹ‘ allosztĂ©rikus jeltovábbĂtĂł hálĂłzat azonosĂtását, az enzimaktivitás rendhagyĂł hĹ‘mĂ©rsĂ©kletfĂĽggĂ©sĂ©nek a konformáciĂłs flexibilitás alapján törtĂ©nĹ‘ Ă©rtelmezĂ©sĂ©t a izopropilmalát dehidrogenáz esetĂ©ben, átmeneti zĂłna felfedezĂ©sĂ©t a rendezett Ă©s rendezetlen szerkezetet kĂłdolĂł aminĂłsav szekvencák között. A komplement fehĂ©rjĂ©k Ă©s funkcionális komplexeik, a flagelláris rendszerek, multidomĂ©n enzimek egyĂĽttes vizsgálata lehetĹ‘vĂ© tette a fehĂ©rjĂ©k önszervezĹ‘dĂ©sĂ©vel, a molekuláris szintű felismerĂ©ssel Ă©s az allosztĂ©rikus jeltovábbĂtás mechanizmusával kapcsolatos általános következtetĂ©sek levonását. | We have determined the structure of C1-Inhibitor and four complement proteinases: C1r, MASP1, MASP2 in zymogen form and MASP2 in activated form. Based on our structural and functional studies we concluded a mechanistic model for the activation of the lectin pathway of the complement system. We also devised a structure based model for the heparin potentiation of C1-Inhibitor. An intrinsically disordered sequence of the bacterial flagellin protein was identified as an export signal (patented). Other significant achievements: the mapping of an allosteric network involved in the ligand induced hinge closure of phosphoglycerate kinase, the interpretation of the odd temperature dependence in the catalytic activity of isopropylmalate dehydrogenase in terms of concerted conformational fluctuations, discovery of the twilight zone between amino acid sequences encoding ordered and disordered conformations. Our coherent studies on the functional protein complexes of the complement system, on flagellar systems, multidomain enzymes enabled us to make some general conclusions regarding the self assembly, recognition and allosteric behaviour of proteins and protein complexes
Fehérjék konformációs dinamikája mint a biomolekuláris felismerés és jelátvitel meghatározó eleme = Protein conformational dynamics as a key determinant in biomolecular recognition and signal transmission
A tĂ©rszerkezet alapján, a konformáciĂłs dinamika figyelembevĂ©telĂ©vel kĂsĂ©reltĂĽk meg az intramolekuláris Ă©s molekulák közötti jelátvitel megĂ©rtĂ©sĂ©t atomi felbontással. KĂsĂ©rleti objektumok: a komplement rendszer, azon belĂĽl is a nemrĂ©g felfedezett lektin Ăşt fehĂ©rjekomplexei, a flagelláris exportrendszer valamint moduláris monomer, dimer Ă©s oligomer felĂ©pĂtĂ©sű enzimek álltak. MegállapĂtottuk, hogy FliI ATPáz, amely kĂ©pes az exportálandĂł fehĂ©rjĂ©je kitekerĂ©sĂ©re, a FliJ, FliH Ă©s FliS komponensekkel egyĂĽtt kĂ©pez olyan szupramolekuláris komplexet, amely kĂ©pes az export szubsztrátumok felismerĂ©sĂ©re. LeĂrtuk a foszfoglicerát kináz enzim alloszterikus működĂ©si mechanizmusát, atomi felbontással. Feltártuk az izopropilmalát dehidrogenáz molekuláris csuklĂłinak működĂ©sĂ©t Ă©s szerepĂ©t az alegysĂ©gek kölcsönhatásaiban. SzelektĂv inhibitorokkal a tankönyvi tĂ©zissel ellentĂ©tes felismerĂ©sre jutottunk, miszerint a komplement rendszer lektin Ăştjának meghatározĂł aktivátora a MASP-1 szerin proteáz. ĂŤgy a komplement aktiválással összefĂĽggĹ‘ betegsĂ©gek Ăşj gyĂłgyszercĂ©lpont molekuláját azonosĂtottuk. FelfedeztĂĽk, hogy a MASP-1 kĂ©pes a kininogĂ©n hasĂtása Ăştján, bradikinint felszabadĂtva, komplement fĂĽggĹ‘ gyulladást keltĂ©sĂ©re. FelfedeztĂĽk, hogy a trombinhoz hasonlĂłan a MASP-1, PAR-4 receptoron keresztĂĽl endotĂ©l sejteket aktivál. BizonyĂtĂ©kot találtunk arra, hogy a fehĂ©rjĂ©k konformáciĂłs dinamikája meghatározza a szerkezet evolĂşciĂłjának lehetsĂ©ges irányait, több milliárd Ă©ves idĹ‘skálán is. | The CUB2 domain of C1r without calcium has disordered structure. This flexibility, necessary for autocativation of C1r inside the C1 complex, is regulated by calcium. Using MASP-selective inhibitors we proved that, in contrast to the previous textbook picture, MASP-1 is the exclusive activator of MASP-2. Blocking the proteolytic activity of MASP-1 prevents activation of the lectin pathway, therefore MASP-1 is a new target in treating complement related diseases. We solved the structure of the catalytic region of MASP-1. The structure explains the special enzymatic characteristics of this complement protease. We discovered a new, inflammation related function of the complement system: MASP-1 is able to directly activate endothelial cells through cleaving protease activated receptor-4. We discovered that MASP-1 is able to cleave kininogen and liberates bradykinin. In this way MASP-1 can contribute to the local inflammatory reaction triggered by complement activation. The allosteric mechanismnof human PGK has been explored at atomic details. In the dimeric enzyme IPMDH structural and site-directed mutagenesis studies revealed the operation of the two main molecular hinges and their relationship with the subunit interactions. We have shown that conformational motions are linked to protein evolution by producing structural variants that can be evolutionarily stabilized. This process is exemplified by segment-swapped proteins, a new group of proteins discovered by us
High levels of acute phase proteins and soluble 70Â kDa heat shock proteins are independent and additive risk factors for mortality in colorectal cancer
Recently, we reported that high soluble Hsp70 (sHsp70) level was a significant predictor of mortality during an almost 3-year-long follow-up period in patients with colorectal cancer. This association was the strongest in the group of <70-year-old female patients as well as in those who were in a less advanced stage of the disease at baseline. According to these observations, measurement of the serum level of sHsp70 is a useful, stage-independent prognostic marker in colorectal cancer, especially in patients without distant metastasis. Since many literature data indicated that measurement of C-reactive protein (CRP) and other acute phase proteins (APPs) may also be suitable for predicting the mortality of patients with colorectal cancer, it seemed reasonable to study whether the effect of sHsp70 and other APPs are related or independent. In order to answer this question, we measured the concentrations of CRP as well as of other complement-related APPs (C1 inhibitor, C3, and C9) along with that of the MASP-2 complement component in the sera of 175 patients with colorectal cancer and known levels of sHsp70, which have been used in our previous study. High (above median) levels of CRP, C1 esterase inhibitor (C1-INH), and sHsp70 were found to be independently associated with poor patient survival, whereas no such association was observed with the other proteins tested. According to the adjusted Cox proportional hazards analysis, the additive effect of high sHsp70, CRP, and C1-INH levels on the survival of patients exceeded that of high sHsp70 alone, with a hazard ratio (HR) of 2.83 (1.13–70.9). In some subgroups of patients, such as in females [HR 4.80 (1.07–21.60)] or in ≤70-year-old patients [HR 11.53 (2.78–47.70)], even greater differences were obtained. These findings indicate that the clinical mortality–prediction value of combined measurements of sHsp70, CRP, and C1-INH with inexpensive methods can be very high, especially in specific subgroups of patients with colorectal cancer