Investigating proteins 'at work' in a living environment at atomic resolution is a major goal of molecular biology, which has not been achieved even though methods for the three-dimensional (3D) structure determination of purified proteins in single crystals or in solution are widely used. Recent developments in NMR hardware and methodology have enabled the measurement of high-resolution heteronuclear multi-dimensional NMR spectra of macromolecules in living cells (in-cell NMR). Various intracellular events such as conformational changes, dynamics and binding events have been investigated by this method. However, the low sensitivity and the short lifetime of the samples have so far prevented the acquisition of sufficient structural information to determine protein structures by in-cell NMR. Here we show the first, to our knowledge, 3D protein structure calculated exclusively on the basis of information obtained in living cells. The structure of the putative heavy-metal binding protein TTHA1718 from Thermus thermophilus HB8 overexpressed in Escherichia coli cells was solved by in-cell NMR. Rapid measurement of the 3D NMR spectra by nonlinear sampling of the indirectly acquired dimensions was used to overcome problems caused by the instability and low sensitivity of living E. coli samples. Almost all of the expected backbone NMR resonances and most of the side-chain NMR resonances were observed and assigned, enabling high quality (0.96 ångström backbone root mean squared deviation) structures to be calculated that are very similar to the in vitro structure of TTHA1718 determined independently. The in-cell NMR approach can thus provide accurate high-resolution structures of proteins in living environments.

The MutM [formamidopyrimidine DNA glycosylase (Fpg)] protein is a trifunctional DNA base excision repair enzyme that removes a wide range of oxidatively damaged bases (N-glycosylase activity) and cleaves both the 3¢-and 5¢-phosphodiester bonds of the resulting apurinic/apyrimidinic site (AP lyase activity). The crystal structure of MutM from an extreme thermophile, Thermus thermophilus HB8, was determined at 1.9 A Ê resolution with multiwavelength anomalous diffraction phasing using the intrinsic Zn 2+ ion of the zinc ®nger. MutM is composed of two distinct and novel domains connected by a¯exible hinge. There is a large, electrostatically positive cleft lined by highly conserved residues between the domains. On the basis of the three-dimensional structure and taking account of previous biochemical experiments, we propose a DNA-binding mode and reaction mechanism for MutM. The locations of the putative catalytic residues and the two DNA-binding motifs (the zinc ®nger and the helix±two-turns±helix motifs) suggest that the oxidized base is¯ipped out from double-stranded DNA in the binding mode and excised by a catalytic mechanism similar to that of bifunctional base excision repair enzymes. Keywords: base excision repair/crystal structure/extreme thermophile/MutM protein/Thermus thermophilus HB8 IntroductionIn aerobic organisms, cellular DNA is frequently damaged by activated oxygen species from aerobic energy metabolism or oxidative stress. Highly reactive oxygen accelerates the rate of spontaneous mutation and has, therefore, been implicated as a causative agent for ageing and in the pathogenesis of disease, including cancer (Breimer, 1990;Ames et al., 1995). In particular, the 8-oxoguanine (GO) lesion is one of the most stable products of oxidative DNA damage (Dizdaroglu, 1985). GO can form a pair with adenine as well as with cytosine, resulting in a G:C to T:A transversion (Wood et al., 1990;Shibutani et al., 1991). In Escherichia coli, the GO repair system, composed of the MutM protein [EC 3.2.2.23], also called formamidopyrimidine DNA glycosylase (Fpg), accompanied by MutY and MutT, prevents this mutation (Michaels et al., 1992).The mutM (fpg) gene encoding the MutM protein is highly conserved across a wide range of aerobic bacteria. These enzymes (M r 30 kDa) possess the invariant N-terminal sequence Pro-Glu-Leu-Pro-Glu-Val-, two strictly conserved lysine residues (Lys52 and Lys147) and a zinc ®nger motif (-Cys-X 2 -Cys-X 16 -Cys-X 2 -Cys-) at the C-terminus ( Figure 1C).MutM was originally isolated from E.coli and characterized as a DNA glycosylase that removes 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (Fapy), the imidazole ring-open form of N 7 -methylguanine (Boiteux et al., 1987). More recently, MutM was reported to bind to a wide range of oxidatively damaged bases (see Figure 5C), including GO paired to cytosine (Tchou et al., 1991), formamidopyrimidine (FapyG or FapyA) and 5-hydroxycytosine (5OHC) (Hatahet et al., 1994) and to apurinic/apyrimidinic (AP) sites (Castaing et al., 1992). MutM h...

Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. In order to complement the existing protocols, and to extend the range of possible applications, we introduce a novel approach for observing in-cell NMR spectra using the sf9 cell/baculovirus system. High-resolution 2D (1)H-(15)N correlation spectra were observed for four model proteins expressed in sf9 cells. Furthermore, 3D triple-resonance NMR spectra of the Streptococcus protein G B1 domain were observed in sf9 cells by using nonlinear sampling to overcome the short lifetime of the samples and the low abundance of the labeled protein. The data were processed with a quantitative maximum entropy algorithm. These were assigned ab initio, yielding approximately 80% of the expected backbone NMR resonances. Well-resolved NOE cross peaks could be identified in the 3D (15)N-separated NOESY spectrum, suggesting that structural analysis of this size of protein will be feasible in sf9 cells.

RAD51, an essential eukaryotic DNA recombinase, promotes homologous pairing and strand exchange during homologous recombination and the recombinational repair of double strand breaks. Mutations that up- or down-regulate RAD51 gene expression have been identified in several tumors, suggesting that inappropriate expression of the RAD51 activity may cause tumorigenesis. To identify chemical compounds that affect the RAD51 activity, in the present study, we performed the RAD51-mediated strand exchange assay in the presence of 185 chemical compounds. We found that 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) efficiently inhibited the RAD51-mediated strand exchange. DIDS also inhibited the RAD51-mediated homologous pairing in the absence of RPA. A surface plasmon resonance analysis revealed that DIDS directly binds to RAD51. A gel mobility shift assay showed that DIDS significantly inhibited the DNA-binding activity of RAD51. Therefore, DIDS may bind near the DNA binding site(s) of RAD51 and compete with DNA for RAD51 binding.