Scientific Report 2005

Molecular Biology

Nuclear Magnetic Resonance in Structural Biology and Structural Genomics

K. Wüthrich, M. Almeida, L. Columbus, T. Etezady, M. Geralt, S. Hiller, R. Horst, M. Johnson, W.J. Placzek, W. Peti, P. Serrano

Members of our laboratory participate in the Joint Center for Structural Genomics (JCSG), the JCSG Center for Innovative Membrane Protein Technologies, and the Functional and Structural Proteomics Analysis of SARS-CoV–Related Proteins Consortium. As part of these studies on protein structure, we develop and use nuclear magnetic resonance (NMR) methods to screen recombinant protein preparations for folded proteins. We are also exploring the use of microcoil NMR equipment combined with microexpression of proteins. We also use NMR spectroscopy to determine the structure of selected proteins from the proteomes under study in the structural genomics programs. Some of our research is described in the following sections.

NMR Screening of Thermotoga Maritima Membrane Proteins

A total of 45 predicted α-helical membrane proteins from Thermotoga maritima were selected as potential targets for solution NMR structural studies. These proteins have between 1 and 4 predicted helical transmembrane segments and have molecular weights less than 16 kD. Of the 45 targets, 10 were overexpressed in Escherichia coli, and 8 of these 10 localized to the bacterial membrane. These 8 protein targets were purified and screened to determine efficient detergents for solubilization.

To evaluate the fold and the aggregation state of the proteins in the best conditions thus identified, we used 1-dimensional ¹H NMR spectroscopy to screen the targets. For 3 of the 8 proteins, the NMR spectra indicated soluble protein-detergent complexes. The transverse relaxation optimized spectroscopy correlation spectra of these 3 targets provided evidence that these 3 proteins are folded helical proteins. Experiments are under way for NMR assignment and structure determination of these α-helical membrane proteins in mixed micelles with detergents

Structure Determinations of Conserved Hypothetical Proteins from T Maritima

The NMR structure of the conserved hypothetical protein TM1816 from T maritima has an α/β topology with 3 α-helices and a 5-stranded β-sheet. The molecular architecture of TM1816 is similar to that of 2 other conserved hypothetical proteins, TM1290 from T maritima (33% sequence identity) and MTH1175 from Methanobacterium thermoautotrophicum (30% sequence identity). These 3 proteins belong to the cluster of orthologous groups 1433 and are structurally similar to the Azobacter vinelandii iron, molybdenum cofactor-binding protein NafY. TM1816 is unique among the 3 homologs because it contains a histidine residue corresponding to the one that is crucial for cofactor binding in NafY.

TM0487 is a 104-residue protein from T maritima that was identified via NMR screening as a potential target for NMR structure determination. The 3-dimensional structure of TM0487 provides a foundation for functional studies of an entire class of proteins, because TM0487 has a large number of homologs on the amino acid sequence level, including 216 nonredundant sequences that contain a type 59 domain of unknown function. So far, a 3-dimensional structure has not been determined for any of these homologous proteins. The conserved residues among the aforementioned 216 sequences are clustered in the hydrophobic core of the TM0487 fold and in a putative active site exposed to the solvent. Overall, strong evidence indicates that the TM0487 fold is preserved in all of this class of domains of unknown function, so that this structure determination provides a foundation for focused functional studies of a wide variety of otherwise so far only minimally characterized proteins.

NMR Studies of an Acyl Carrier Protein from the Cyanobacterium Anabaena

Asl1650, a protein obtained from the cyanobacterium Anabaena, was identified as an ortholog of a mouse protein domain as part of a JCSG bioinformatics strategy to extend information on the protein folding space of eukaryotic proteins. The protein was selected for NMR structure determination on the basis of an NMR screen of recombinant mouse protein homologs expressed in E coli.

Acyl carrier proteins (ACPs) are central components of complex multienzyme systems that function in the metabolism of all living organisms. These systems catalyze the biosynthesis of fatty acids, signaling molecules, and bioactive natural products. The polyketide synthases and nonribosomal peptide synthetases of microorganisms produce compounds with antibiotic and anticancer activities. An understanding of structure-function relationships in these widely distributed enzyme systems is thus of obvious interest for the design of new therapeutic compounds.

The protein Asl1650 is only distantly related to previously characterized ACPs. It was derived from Anabaena sp PCC 7120, a filamentous cyanobacterium. Members of this genus of cyanobacteria produce a variety of bioactive compounds, which are as yet only poorly characterized. We determined the solution structure of Asl1650 by using high-resolution NMR spectroscopy. The structure had a surprising similarity to the structures of peptidyl carrier protein domains, which usually occur as single domains of giant, multifunctional proteins. A variant active-site sequence, asparagine–serine–serine, occurs in similar orientation to the aspartic acid–serine–leucine sequence of known ACPs. These structural similarities suggest that Asl1650 may function as a discrete peptidyl carrier protein domain in a nonribosomal peptide synthetase pathway or a hybrid polyketide synthase–nonribosomal peptide synthetase pathway.

Publications

Almeida, M.S., Peti, W., Wüthrich, K. ¹H-, ¹³C- and ¹⁵N-NMR assignment of the conserved hypothetical protein TM0487 from Thermotoga maritima. J. Biomol. NMR 29:453, 2004.

Etezady-Esfarjani, T., Herrmann, T., Peti, W., Klock, H.E., Lesley, S.A., Wüthrich, K. NMR structure determination of the hypothetical protein TM1290 from Thermotoga maritima using automated NOESY analysis. J. Biomol. NMR 29:403, 2004.

Page, R., Peti, W., Wilson, I.A., Stevens, R.C., Wüthrich, K. NMR screening and crystal quality of bacterially expressed prokaryotic and eukaryotic proteins in a structural genomics pipeline. Proc. Natl. Acad. Sci. U. S. A. 102:1901, 2005.

Peti, W., Etezady-Esfarjani, T., Herrmann, T., Klock, H.E., Lesley, S.A., Wüthrich, K. NMR for structural proteomics of Thermotoga maritima: screening and structure determination. J. Struct. Funct. Genomics 5:205, 2004.

Peti, W., Norcross, J., Eldridge, G., O’Neil-Johnson, M. Biomolecular NMR using a microcoil NMR probe: new technique for the chemical shift assignment of aromatic side chains in proteins. J. Am. Chem. Soc. 126:5873, 2004.