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Linker Peptide (linker + peptide)
Selected AbstractsHeme-hemopexin: A ,Chronosteric' heme-proteinIUBMB LIFE, Issue 11 2007Paolo Ascenzi Abstract Hemopexin (HPX) serves as scavenger and transporter of toxic plasma heme to the liver. HPX is formed by two four-bladed ,-propeller domains, resembling two thick disks that lock together at a 90° angle. The heme is bound between the two ,-propeller domains in a pocket formed by the interdomain linker peptide. Residues His213 and His266 coordinate the heme iron atom giving a stable bis-histidyl complex. The HPX-heme geometry is reminiscent of heme-proteins endowed with ligand binding and (pseudo-)enzymatic properties. HPX-heme binds reversibly CO, ,NO, and cyanide by detaching His213; however, O2 induces HPX-heme(II) oxidation. Furthermore, HPX-heme(II) facilitates ,NO/O2 and ,NO/peroxynitrite scavenging. Heme sequestering by HPX prevents heme-mediated activation of oxidants which induce the low-density lipoprotein oxidation. Here, ligand binding and (pseudo-)enzymatic properties of HPX-heme are reviewed. HPX, acting not only as a heme carrier but also displaying transient heme-based ligand binding and (pseudo-)enzymatic properties, could be considered a ,chronosteric' heme-protein. IUBMB Life, 59: 700-708, 2007 [source] Structure of cyclized green fluorescent proteinACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2002Andreas Hofmann Crystals of cyclic green fluorescent protein (cGFP) engineered by the previously reported split intein technology [Iwai et al. (2001), J. Biol. Chem.276, 16548,16554] were obtained and the structure was solved using molecular replacement. Although the core of the protein can unambiguously be fitted from the first to the last residue of the genuine sequence, the electron density in the region of the linker peptide is rather poor owing to the high water content of the crystals. Therefore, it is concluded that this part of the protein is highly disordered in the present structure and is very flexible. This is supported by the absence of crystal contacts in the linker-peptide region and the fact that the core of the protein exhibits a very similar conformation to that known from other GFP structures, thereby not implicating any constraints arising from the presence of the artificial linker. Nevertheless, the density is consistent with the loop being intact, as confirmed by mass spectroscopy of dissolved crystals. The present structure contains an antiparallel cGFP dimer where the dimer interface is clearly different from other crystal structures featuring two GFP molecules. This adds further support to the fact that the cylinder surface of GFP is rather versatile and can employ various polar and non-polar patches in protein,protein interactions. [source] Hemopexin: The primary specific carrier of plasma heme,BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 5 2002Marco Mattu Abstract Hemopexin (HPX) is the primary specific carrier of plasma heme and participates in its clearance by transport to the liver. After delivering the heme intracellularly, HPX is released intact into the bloodstream. HPX is formed by two four-bladed ,-propeller domains, resembling two thick disks that lock together at a 90° angle; the face of the N-terminal ,-propeller domain packs against one edge of the C-terminal domain. Each propeller blade comprises a four-stranded antiparallel ,-sheet, with the first and the fourth blades tied together by disulfide bridges. The heme ligand is bound between the two four-bladed ,-propeller domains in a pocket formed by the interdomain linker peptide. Residues His-213 and His-266 coordinate the heme iron atom giving a stable bis-histidyl Fe(III) complex. Heme release results from opening of the heme binding pocket, through movement of the two ,-propeller domains and/or the interdomain linker peptide. [source] Cloning, expression, and identification of anti-carbofuran single chain Fv geneBIOTECHNOLOGY PROGRESS, Issue 4 2009Hong Wang Abstract Phage display method was used to clone anti-carbofuran (CBF) single chain Fv (scFv) gene. The heavy chain and light chain variable region genes were amplified by the polymerase chain reaction from the CBF-specific hybridoma cell lines 5D3 and assembled as a scFv DNA fragment with linker peptide (Gly4Ser)3. The scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti-CBF antibody libraries were then constructed. After one round of panning with CBF-ovalbumin (CBF-OVA) as a conjugate, antigen-binding positive recombinant phage clones were successfully selected by enzyme-linked immunosorbent assay (ELISA). The positive phages were used to infect Escherichia coli HB2151 cells and the expression of the soluble scFv antibodies was then induced by IPTG. The scFv antibody was about 31 kDa by SDS-PAGE and showed HRP-anti-E-tag antibody-recognized activity by Western blotting. The indirect competitive ELISA (icELISA) showed that the recombinant scFv antibody could competitively combine with CBF, with the IC50 value of 1.07 ng/mL. The cross reactivity studies showed that the anti-CBF scFv antibody, similar to the parent monoclonal antibody, poses high specificity to CBF and has little reactivity to the analogs. Taken together, these findings suggest that the recombinant scFv antibody can be used for further developing immunoassay method for CBF. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Large-scale expression and thermodynamic characterization of a glutamate receptor agonist-binding domainFEBS JOURNAL, Issue 13 2000Dean R. Madden The ionotropic glutamate receptors (GluR) are the primary mediators of excitatory synaptic transmission in the brain. GluR agonist binding has been localized to an extracellular domain whose core is homologous to the bacterial periplasmic binding proteins (PBP). We have established routine, baculovirus-mediated expression of a complete ligand-binding domain construct at the 10-L scale, yielding 10,40 milligrams of purified protein. This construct contains peptides that lie outside the PBP-homologous core and that connect the domain core to the transmembrane domains of the channel and to the N-terminal ,X'-domain. These linker peptides have been implicated in modulating channel physiology. Such extended constructs have proven difficult to express in bacteria, but the protein described here is stable and monomeric. Isothermal titration calorimetry reveals that glutamate binding to the domain involves a substantial heat capacity change and that at physiological temperatures, the reaction is both entropically and enthalpically favorable. [source] Strategy for selecting and characterizing linker peptides for CBM9-tagged fusion proteins expressed in Escherichia coliBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2007Mojgan Kavoosi Abstract The influence of linker design on fusion protein production and performance was evaluated when a family 9 carbohydrate-binding module (CBM9) serves as the affinity tag for recombinant proteins expressed in Escherichia coli. Two bioinformatic strategies for linker design were applied: the first identifies naturally occurring linkers within the proteome of the host organism, the second involves screening peptidases and their known specificities using the bioinformatics software MEROPSÔ to design an artificial linker resistant to proteolysis within the host. Linkers designed using these strategies were compared against traditional poly-glycine linkers. Although widely used, glycine-rich linkers were found by tandem MS data to be susceptible to hydrolysis by E. coli peptidases. The natural (PT)xP and MEROPSÔ-designed S3N10 linkers were significantly more stable, indicating both strategies provide a useful approach to linker design. Factor Xa processing of the fusion proteins depended strongly on linker chemistry, with poly(G) and S3N10 linkers showing the fastest cleavage rates. Luminescence resonance energy transfer studies, used to measure average distance of separation between GFP and Tb(III) bound to a strong calcium-binding site of CBM9, revealed that, for a given linker chemistry, the separation distance increases with increasing linker length. This increase was particularly large for poly(G) linkers, suggesting that this linker chemistry adopts a hydrated, extended configuration that makes it particularly susceptible to proteolysis. Differential scanning calorimetry studies on the PT linker series showed that fusion of CBM9 to GFP did not alter the Tm of GFP but did result in a destabilization, as seen by both a decrease in Tm and ,Hcal, of CBM9. The degree of destabilization increased with decreasing length of the (PT)xP linker such that ,Tm,=,,8.4°C for the single P linker. Biotechnol. Bioeng. 2007;98: 599,610. © 2007 Wiley Periodicals, Inc. [source] | |||||||||||||