Uncovering the role of the flexible C-terminal tail: A model study with Strep-tagged GFP.

Recently, it has been recognized that, much like an electric current in an electric circuit, dynamic disruptions from flexible, unstructured regions distal to the active region are transferred through the contact network to the active site and influence protein stability and/or function. As transmembrane proteins frequently possess the β-barrel structure, studies of proteins with this topology are required. The unstructured lid segments of the β-barrel GFP protein are conserved and could play a role in the backbone stabilization required for chromophore function.

Expression and assembly of active human cardiac troponin in Escherichia coli.

Cardiomyopathy-related mutations in human cardiac troponin subunits, including troponin C (hcTnC), troponin I (hcTnI), and troponin T (hcTnT), are well-documented. Recently, it has been recognised that human cardiac troponin (hcTn) is a sophisticated allosteric system. Therefore, the effect of drugs on this protein complex should be studied with assembled hcTn rather than a short fragment of a subunit or the subunit itself.

Firm wheat-germ cell-free system with extended vector usage for high-throughput protein screening.

The wheat germ cell-free system is composed out of five basic steps, growth of Escherichia coli harboring plasmid, first colony-PCR, second PCR, transcription, and translation. Improvements of culture medium, colony based PCR, and modifications within the split primer set of the second PCR amplify both DNA and RNA levels. This yields more than 5 times increase in protein amount for pEU-originated templates.

Defective dynamic properties of human cardiac troponin mutations.

Mutations in Troponin I (TnI) and Troponin T (TnT) are closely linked to familial hypertrophic cardiomyopathy (FHC) and hypertrophic cardiomyopathy (HCM), but the underlying molecular mechanism is not yet well understood. There might be a close link between the defective dynamic properties and the functional aberrations of hcTroponin. To prove this hypothesis, we undertook detailed NMR relaxation measurements of [(2)H, (13)C, (15)N] labeled proteins reconstituted into hcTroponin in both the Ca(2+)- and the Mg(2+)-loaded state.

The use of high-pressure nuclear magnetic resonance to study protein folding.

Recent development of high-pressure cells for a variety of spectroscopic methods has enabled the use of pressure as one of the commonly used perturbations along with temperature and chemical perturbations to study folding/unfolding reactions of proteins. Although various high-pressure spectroscopy techniques have their own significance, high-pressure nuclear magnetic resonance (NMR) is unique in that it allows one to gain residue-specific and atom-detailed information from proteins under pressure. Furthermore, because of a peculiar volume property of a protein, high-pressure NMR allows one to obtain structural information of a protein in a wide conformational space from the bottom to the upper region of the folding funnel, giving structural reality for the "open" state of a protein proposed from hydrogen exchange.

Effects of melanogenesis-inducing nitric oxide and histamine on the production of eumelanin and pheomelanin in cultured human melanocytes.

Melanin pigments produced in human melanocytes are classified into two categories; black coloured eumelanin and reddish-yellow pheomelanin. Stimulation of melanocytes with alpha-melanocyte-stimulating hormone (alpha-MSH), one of several melanogenic factors, has been reported to enhance eumelanogenesis to a greater degree than pheomelanogenesis, which contributes to hyperpigmentation in skin. Nitric oxide (NO) and histamine are also melanogenesis-stimulating factors that are released from cells surrounding melanocytes following ultraviolet (UV) irradiation.

Pressure-induced unfolding of the molten globule of all-Ala alpha-lactalbumin.

Pressure-induced unfolding of a molten globule (MG) was studied in a residue-specific manner with (1)H-(15)N two-dimensional NMR spectroscopy using a variant of human alpha-lactalbumin (alpha-LA), in which all eight cysteines had been replaced with alanines (all-Ala alpha-LA). The NMR spectrum underwent a series of changes from 30 to 2000 bar at 20 degrees C and from -18 degrees C to 36 degrees C at 2000 bar, showing a highly heterogeneous unfolding pattern according to the secondary structural elements of the native structure. Unfolding began in the loop part of the beta-domain, and then extended to the remainder of the beta-domain, after which the alpha-domain began to unfold.