ABSTRACT
ß-trefoil fold, consisting of a six stranded ß-barrel capped at one end by a lid comprising of another six ß-strands, is one of the most important folds among proteins. Important classes of proteins like Interleukins (ILs), Fibroblast Growth Factors (FGFs), Kunitz (STI) family of inhibitors etc. belong to this fold. Their core is packed by hydrophobic residues contributed by the 6 stranded ß-barrel and three ß-hairpins that make essential contacts with each other and keep the protein in 'topologically minimal frustrated state'. A complete database analysis of the core residues of the ß-trefoil fold proteins presented here identified a conserved tryptophan (W91) residue in the Kunitz (STI) family of inhibitors that projects from the lid and interacts with the bottom layer residues of the barrel. This kind of interactions is unique in Kunitz (STI) family because no other families of ß-trefoil fold have such a shear sized residue at the barrel lid junction; suggesting its possible importance in packing and stability. We took WCI as a representative of this family and prepared four cavity creating mutants W91F-WCI, W91M-WCI, W91I-WCI & W91A-WCI. CD experiments show that the secondary structure of the mutants remains indistinguishable with the wild type. Crystal structures of the mutants W91F-WCI, W91M-WCI & W91A-WCI also show the same feature. However, slight readjustments of the side chains around the site of mutation have been observed so as to minimize the cavity created due to mutation. Comparative stability of these mutants, estimated using heat denaturation CD spectroscopy, indicates that stability of the mutants inversely correlates with the size of the cavity inside the core. Interestingly, although we mutated at the core, mutants show varying susceptibility against tryptic digestion that grossly follow their instability determined by CD. Our findings suggest that the W91 residue plays an important role in determining the stability and packing of the core of WCI.
Subject(s)
Protein Structure, Secondary , Protein Structure, Tertiary , Trypsin Inhibitor, Kunitz Soybean/chemistry , Tryptophan/chemistry , Amino Acids/chemistry , Amino Acids/genetics , Amino Acids/metabolism , Circular Dichroism , Crystallography, X-Ray , Fibroblast Growth Factors/chemistry , Fibroblast Growth Factors/metabolism , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Interleukins/chemistry , Interleukins/metabolism , Models, Molecular , Mutation , Protein Binding , Protein Stability , Temperature , Trypsin Inhibitor, Kunitz Soybean/metabolism , Trypsin Inhibitors/chemistry , Trypsin Inhibitors/metabolism , Tryptophan/genetics , Tryptophan/metabolismABSTRACT
Two new alternating µ1,1- and µ1,3-azido bridged chains, [Cu(N3)2(mtn)]n (1) and [Ni(N3)2(mtn)]n (2) where mtn = N-methyl-1,3-propanediamine, have been synthesized and characterized by single crystal X-ray diffraction. In both complexes, each metal atom is coordinated to six nitrogen atoms from four azide anions and one N-methyl-1,3-propanediamine molecule in a distorted octahedral geometry. In 1, the basal-apical double µ1,1-azido bridged Cu(ii)-dimers are connected through two asymmetric µ1,3-N3 bridges to form a 1D chain in which one of the azide acts as a µ1,1,3 bridge while the other one is terminal. The structure of 2 is very similar but one of the azide ions acts as µ1,1 and the other as µ1,3 to form an alternating chain. Magnetic data of both compounds were fitted to an alternating ferro/antiferromagnetic chain model (g = 2.120, JF = +15.6 cm(-1), JAF = -2.6 cm(-1) and a paramagnetic impurity of ca. 0.4% for 1 and g = 2.180, JF = +23 cm(-1) and JAF = -33 cm(-1) for 2).
