Recent advances have fundamentally changed the ways in which synthetic amino acids are incorporated into proteins, enabling their efficient and multiple-site incorporation, in addition to the 20 canonical amino acids. This development provides opportunities for fresh approaches toward addressing fundamental problems in bioengineering. In the present study, we showed that the structural stability of proteins can be enhanced by integrating bulky halogenated amino acids at multiple selected sites. Glutathione S-transferase was thus stabilized significantly (by 5.2 and 5.6 kcal/mol) with 3-chloro- and 3-bromo-l-tyrosines, respectively, incorporated at seven selected sites. X-ray crystallographic analyses revealed that the bulky halogen moieties filled internal spaces within the molecules, and formed non-canonical stabilizing interactions with the neighboring residues. This new mechanism for protein stabilization is quite simple and applicable to a wide range of proteins, as demonstrated by the rapid stabilization of the industrially relevant azoreductase.
The circadian clock controls daily rhythms in many physiologic processes, and the clock oscillation is regulated by external time cues such as light, temperature, and feeding. In mammals, the transcriptional regulation of clock genes underlies the clock oscillatory mechanism, which is operative even in cultured fibroblasts. We previously demonstrated that glucose treatment of rat-1 fibroblasts evokes circadian expression of clock genes with a rapid induction of Tieg1 transcript encoding a transcriptional repressor. Here, we found diurnal variation of both Tieg1 mRNA and nuclear TIEG1 protein levels in the mouse liver with their peaks at day ⁄ night transition and midnight, respectively. In vitro experiments showed that TIEG1 bound to Bmal1 gene promoter and repressed its transcriptional activity through two juxtaposed GC boxes near the transcription initiation site. The GC box ⁄ TIEG1-mediated repression of Bmal1 promoter was additive to RORE-dependent repression by REV-ERBa, a well-known repressor of Bmal1 gene. In cell-based real-time assay, siRNA-mediated knock-down of TIEG1 caused period shortening of cellular bioluminescence rhythms driven by Bmal1-luciferase and Per2-luciferase reporters. These findings highlight an active role of TIEG1 in the normal clock oscillation and GC box-mediated regulation of Bmal1 transcription.
Galectins are a group of animal lectins defined by their binding specificity for b-galactosides and by an evolutionarily conserved sequence motif in their carbohydrate-recognition domains. Galectins are involved in a wide variety of biological phenomena, including development, cell differentiation, tumor metastasis, apoptosis, RNA splicing, and regulation of immune function. [3][4][5][6][7][8] Galectins can be classified into three types based on their molecular architecture: proto-type, chimera-type, and tandem-repeat type. LEC-1, the 32-kDa galectin of the nematode Caenorhabditis elegans, was the first example of a tandem repeat-type galectin composed of two homologous regions. 9,10) Using a reinforced frontal affinity chromatography system, [11][12][13] we demonstrated that individual galectins have acquired a unique sugar-binding specificity. Thus, while all galectins recognize the basic disaccharide units (Galb1-4(3)Glc(NAc)), individual galectins show enhanced affinity for either "branched", "repeated", or "substituted" glycans, presumably resulting in specialized biological functions. For example, galectin-3 has been shown to have a high affinity for blood group A saccharide (GalNAca1-3(Fuca1-2)Galb1-3GlcNAc), 14,15) whereas other galectins, such as galectin-1, lack this feature. Although the eight highly conserved amino acid residues in galectins are known to be important for the recognition of the basic disaccharide unit, [16][17][18][19][20][21] there is little information concerning which other amino acid residues might contribute to specific sugar recognition. According to several X-ray crystallographic studies of galectins, [19][20][21] the highly conserved amino acid residues that form sugar-binding sites for Galb1-4(3)Glc(NAc) are all located in the three b-strands S4-S6 in the six-stranded bsheet (b-strands S1-S6) facing the bound sugar. Amino acid residue(s) responsible for specific sugar recognition may exist in b-strands other than S4-S6, and identification of these residues should help elucidate the binding mechanisms and biological functions of galectins.In a previous study, we analyzed the detailed sugar-binding properties of the two domains of the 32-kDa tandem repeat-type galectin LEC-1 from the nematode C. elegans using frontal affinity chromatography. 22) We showed that the isolated N-terminal lectin domain of LEC-1 (LEC-1N) has a high binding affinity for (GalNAca1-3(Fuca1-2)Galb1-3GlcNAcb1-3Galb1-4Glc-PA) whereas the isolated C-terminal lectin domain (LEC-1C) has a very low binding affinity for this sugar. In contrast, Fuca1-2Galb1-3GlcNAcb1-3Galb1-4Glc-PA (H Antigen) was recognized by both domains.Upon consideration of the X-ray crystal structure of LEC-1 in complex with galactose, 23) as well as the X-ray crystal structures of other galectins, we predicted that several nonconserved amino acids in LEC-1N may modulate specific sugar binding. These amino acids are located on the bstrands facing the sugar-binding site and would be close enough to GalNAca1-3 and Fuca1-2 structures attach...
A 16 kDa buckwheat protein (BWp16) is a major allergen responsible for immediate hypersensitivity reactions including anaphylaxis. A deletion mutant of BWp16 (rBWp16ÁN) was overproduced and purified and was shown to be immunologically active. A three-wavelength MAD data set was collected from a crystal of selenomethionine-labelled rBWp16ÁN. The crystal belonged to the triclinic space group P1, with unit-cell parameters a = 28.39, b = 31.54, c = 32.20 Å , = 111.92, = 108.91, = 98.74 . One monomer was expected to be present in the asymmetric unit based on the calculated Matthews coefficient of 1.76 Å 3 Da À1 .
Galectin LEC-1 isolated from the nematode Caenorhabditis elegans was the first galectin found in invertebrates and also the first tandem-repeat-type galectin identified, containing two homologous carbohydrate-binding sites. This galectin is localized most abundantly in the adult cuticle and possibly plays a role in the formation of epidermal layers. We succeeded in crystallizing LEC-1 composed of 279 amino acids with a calculated molecular weight of 31,809 Da under two independent sets of conditions as a result of extensive screening. The crystals grown under one set of conditions belong to the triclinic space group P1, with unit-cell parameters a = 48.44, b = 52.13, c = 64.24 A, alpha = 108.73, beta= 91.39, and gamma = 98.45 degrees and two protein molecules per unit cell. The crystals grown under the other set of conditions which included lactose belong to the monoclinic space group P2(1), with unit-cell parameters a = 52.90, b = 47.01, c = 66.16 A, and beta= 113.30 degrees and one protein molecule per asymmetric unit.
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