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Toward the functional oligomerization state of tryptophan-rich sensory proteins.

ABSTRACT: A conserved family of tryptophan-rich sensory proteins (TspO) mediates the transport of heme degradation intermediates across membranes. In eukaryotes, the homologous mitochondrial translocator protein (TSPO) binds cholesterol and radioligands as monomer. On the basis of the mammalian TSPO structure, bioinformatic analysis, and a 10 Å resolution electron microscopy map of TspO from Rhodobacter sphaeroides, we developed a model of the tertiary and quaternary structure of TspO that is in agreement with available mutagenesis data. Our study provides insight into the conformational basis for the restricted interaction of bacterial TspO with radioligands and the functional oligomerization state of bacterial TspO proteins.

SUBMITTER: Jaremko L

PROVIDER: S-EPMC4116663 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Toward the functional oligomerization state of tryptophan-rich sensory proteins.

Jaremko Lukasz L Jaremko Mariusz M Becker Stefan S Zweckstetter Markus M

Protein science : a publication of the Protein Society 20140522 8

A conserved family of tryptophan-rich sensory proteins (TspO) mediates the transport of heme degradation intermediates across membranes. In eukaryotes, the homologous mitochondrial translocator protein (TSPO) binds cholesterol and radioligands as monomer. On the basis of the mammalian TSPO structure, bioinformatic analysis, and a 10 Å resolution electron microscopy map of TspO from Rhodobacter sphaeroides, we developed a model of the tertiary and quaternary structure of TspO that is in agreement ...[more]

PMID: 24817333

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Project description:BACKGROUND:Conventional fluorescent proteins, such as GFP, its derivatives and flavin mononucleotide based fluorescent proteins (FbFPs) are often used as fusion tags for detecting recombinant proteins during cultivation. These reporter tags are state-of-the-art; however, they have some drawbacks, which can make on-line monitoring challenging. It is discussed in the literature that the large molecular size of proteins of the GFP family may stress the host cell metabolism during production. In addition, fluorophore formation of GFP derivatives is oxygen-dependent resulting in a lag-time between expression and fluorescence detection and the maturation of the protein is suppressed under oxygen-limited conditions. On the contrary, FbFPs are also applicable in an oxygen-limited or even anaerobic environment but are still quite large (58% of the size of GFP). RESULTS:As an alternative to common fluorescent tags we developed five novel tags based on clustered tryptophan residues, called W-tags. They are only 5-11% of the size of GFP. Based on the property of tryptophan to fluoresce in absence of oxygen it is reasonable to assume that the functionality of our W-tags is also given under anaerobic conditions. We fused these W-tags to a recombinant protein model, the anti-CD30 receptor single-chain fragment variable antibody (scFv) Ki-4(scFv) and the anti-MucI single-chain fragment variable M12(scFv). During cultivation in Microtiter plates, the overall tryptophan fluorescence intensity of all cultures was measured on-line for monitoring product formation via the different W-tags. After correlation of the scattered light signal representing biomass concentration and tryptophan fluorescence for the uninduced cultures, the fluorescence originating from the biomass was subtracted from the overall tryptophan signal. The resulting signal, thus, represents the product fluorescence of the tagged and untagged antibody fragments. The product fluorescence signal was increased. Antibodies with W-tags generated stronger signals than the untagged construct. CONCLUSIONS:Our low-molecular-weight W-tags can be used to monitor the production of antibody fragments on-line. The binding specificity of the recombinant fusion protein is not affected, even though the binding activity decreases slightly with increasing number of tryptophan residues in the W-tags. Thus, the newly designed W-tags offer a versatile and generally applicable alternative to current fluorescent fusion tags.

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Toward the functional oligomerization state of tryptophan-rich sensory proteins.

Publications

Toward the functional oligomerization state of tryptophan-rich sensory proteins.

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