Biochemical and biophysical characterization of mutant T. durum metallothionein

Abstract

Metallotiyoneinler (MTler) düsük moleküler ağırlıklı, çok sayıda sistein içerenve metal bağlayan proteinlerdir. Metallotiyoneinlerin kesfi yaklasık 60 yıl once atböbreğinde kadmiyum bağlayan protein olarak gerçeklesmistir. (margoshes vallee).MTler pek çok canlıda bulunur ve sınıflandırmaları filojenik yakınlıklarına vesekanslarındaki sistein motiflerinin dağılımına gore olmustur. (binz kagi 2001). MTlerd10 elektron konfigürasyonuna sahip pek çok metali sisteinleriyle koordine edebilirler.(vasak hasler 2000).Bu çalısmadaki amaç Triticum durum MTsinin (dMT) metal bağlamaözelliklerinin sistein sayısı ve motifleriyle olan iliskisini incelemektir. Bunun içindMT'nin 65. glisini sisteine mutagenez metoduyla dönüstürülmüs (G65C) ve mutantdMT E. coli de Glutatyon S-Transferaz (GST) füzyon protein olarak sentezlenmistir(GSTG65C). G65C sentezlenmis, saflastırılmıs ve boyut kromatografisi, SDS- ve doğalpoliakrilamit jel eletroforezi, dairesel dikroizm, absorbans spektroskopisi, dinamik ısıksaçılımı, endüktif çiftlenmis plazma optik ısıma spektroskopisi ve düsük açılı X-Raysaçılımı ile yapısal olarak incelenmistir. Bunun yanında G65C metallerindenarındırılarak apoprotein halindeki yapısı da incelenmistir.

Metallothionein (MT) family is characterized by low molecular weight cysteinerich proteins that bind d10 metals through thiolate bonds (Vasak et al. 2000). MTs arefound in wide range of organisms and their classification was based on the phylogeneticrelationships and patterns of distribution of Cys residues along the MT sequences(Kojima et al. 1999).In this study, the aim was to determine structural and metal binding properties ofmutant Triticum durum metallothionein (G65C) and to analyze differences in metalbinding capacity between the mutant and the native durum metallothionein (dMT). Amutation was introduced into one of the cys motifs (C-X-C) at DNA level to mimic amammalian motif (C-X-C-C). The 65th glycine was mutated to a cysteine and themutated gene was expressed in E. coli as Glutathione S-Transferase (GST) fusionprotein (GSTG65C). G65C was cleaved from GST, purified and demetallated (apo-G65C) for biochemical and biophysical characterization.G65C mutant showed a higher level of oligomerization and polydispersity, butthe cadmium content was 5 Cd++/protein which is similar to that of native dMT.Homogeneous solutions of apo-G65C were used for reconstitution studies. Apoproteinwas reconstituted with cadmium and zinc and changes in structure were monitored byCD and absorbance measurements. Fully cadmium loaded protein was significantlydifferent from holo-G65C purified directly from E.coli. During reconstitution majorchanges were observed at 230 and 250 nm. The strong absorbance increase observed atv230 nm indicates that significant conformational rearrangements take place in the hingeregion - connecting metal binding domains ? as well as within the cys-rich domains.The folding process in vitro takes place in a nonlinear fashion and is different from thatof native dMT with the bridging thiolates forming later. The structural model developedfrom SAXS measurements show that both apo- and holo- G65C have asymmetricstructures, the apoprotein being more elongated (maximum dimension: ~6 nm for holoand~10 nm for apo-G65C). The models are consistent with two cluster structure.Results presented show that metal binding capacity is not dependent only on cysquantity, but also on cys motifs.