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Journal Issue - Volume 2 Issue 9 (September 1993)

Abstract Crystal and NMR structures of helical cytokines–interleukin‐4 (IL‐4), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), and interleukin‐2 (IL‐2)–have been compared. Root mean square deviations in the Cα coordinates for the conserved regions of the helices were 1–2 Å between different cytokines, about twice the differences observed for independently determined crystal and solution structures of IL‐4. Considerable similarity in...

Abstract Pepsin contains, in a single chain, two conformationally homologous lobes that are thought to have been evolutionarily derived by gene duplication and fusion. We have demonstrated that the individual recombinant lobes are capable of independent folding and reconstitution into a two‐chain pepsin or a two‐chain pepsinogen (Lin, X., et al., 1992, J. Biol. Chem. 267, 17257–17263). Pepsin spontaneously inactivates in neutral or alkaline...

Abstract C5a is an inflammatory mediator that evokes a variety of immune effector functions including chemotaxis, cell activation, spasmogenesis, and immune modulation. It is well established that the effector site in C5a is located in the C‐terminal region, although other regions in C5a also contribute to receptor interaction. We have examined the N‐terminal region (NTR) of human C5a by replacing selected residues in the NTR with...

Abstract α‐1‐Proteinase inhibitor (A1Pi) is a monomeric secreted protein glycosylated at asparagines 46, 83, and 247. For this study cDNAs for M (normal) and S (Glu264 → Val) variants of A1Pi were altered by site‐directed mutagenesis to produce the combinations of single, double, and triple mutants that can be generated by changing the codons normally specifying these Asn residues to encode Gln. The fates of the mutant proteins were...

Abstract To help understand the structure/function relationships in antifreeze proteins (AFP), and to define the motifs required for ice binding, a Type III AFP suitable for two‐dimensional (2D) NMR studies was produced in Escherichia coli. A synthetic gene for one of the Type III AFP isoforms was assembled in a T7 polymerase‐directed expression vector. The 67‐amino acid‐long gene product differed from the natural AFP by inclusion of an...

Abstract The structural and functional consequences of replacing Ω‐loop A (residues 18–32) in yeast iso‐1‐cytochrome c with the corresponding loop of Rhodospirillum rubrum cytochrome c2 have been examined. The three‐dimensional structure of this loop replacement mutant RepA2 cytochrome c, and a second mutant RepA2(Val 20) cytochrome c in which residue 20 was back substituted to valine, were determined using X‐ray diffraction techniques. A change in the molecular ...

Abstract Several amino groups of recombinant human erythropoietin are selectively cross‐linked by specific cross‐linkers including disuccinimidyl suberate or dithiobis(succinimidyl propionate). Intramolecular cross‐linkings are obtained without significant change of the protein conformation using appropriate concentrations (0.2 mM) of the cross‐linkers, which possess an 11–12‐Å length of a spacer between two reacting groups....

Abstract Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin‐dependent aromatic amino acid hydroxylases. Comparison of the amino acid sequences of these three proteins shows that the C‐terminal two‐thirds are homologous, while the N‐terminal thirds are not. This is consistent with a model in which the C‐terminal two‐thirds constitute a conserved catalytic domain to...

Abstract In this paper we describe the expression and purification from bacteria of the recombinant basic leucine zipper (bZip) domain of the cAMP response element binding protein, CREB327. The bZip peptide, CREB259–327, purified to near homogeneity, maintains the sequence‐specific CRE site recognition demonstrated by in vitro competition assays. Alkylation of the three cysteine residues of CREB259–327 was employed to prevent aggregation of the...

Abstract Two murine interleukin‐6 (mIL‐6) variants were constructed using the polymerase chain reaction (PCR), one lacking the last five residues (183–187) at the C‐terminus (pMC5) and another with the last five residues of mIL‐6 substituted by the corresponding residues of human IL‐6 (pMC5H). The growth stimulatory activity of pMC5 on the mouse hybridoma cell line 7TD1 was <0.05% of mIL‐6, whereas pMC5H and mIL‐6 were...

Abstract Activated protein C (APC) exerts its physiologic anticoagulant role by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. The synthetic peptide‐(311–325) (KRNRTFVLNFIKIPV), derived from the heavy chain sequence of APC, potently inhibited APC anticoagulant activity in activated partial thromboplastin time (APTT) and Xa‐1‐stage coagulation assays in normal and in protein S‐depleted plasma with 50%...

Abstract Some slow‐folding phases in the in vitro refolding of proteins originate from the isomerization of prolyl‐peptide bonds, which can be accelerated by a class of enzymes called prolyl isomerases (PPIs). We used the in vitro folding of an antibody Fab fragment as a model system to study the effect of PPI on a folding reaction that is only partially reversible. We show here that members of both subclasses of PPIs, cyclophilin...

Abstract Apocytochrome b5 from rabbit liver was studied by scanning calorimetry, limited proteolysis, circular dichroism, second derivative spectroscopy, and size exclusion chromatography. The protein is able to undergo a reversible two‐state thermal transition. However, transition temperature, denaturational enthalpy, and heat capacity change are reduced compared with the holoprotein. Apocytochrome b5 stability in terms of Gibbs energy change...

Abstract A method is presented to model loops of protein to be used in homology modeling of proteins. This method employs the ESAP program of Higo et al. (Higo, J., Collura, V., & Garnier, J., 1992, Biopolymers 32, 33–43) and is based on a fast Monte Carlo simulation and a simulated annealing algorithm. The method is tested on different loops or peptide segments from immunoglobulin, bovine pancreatic trypsin inhibitor, and bovine trypsin....

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