Jens Köditz

Local fluctuations vs. global unfolding of proteins investigated by limited proteolysis

The structure of a protein molecule consists of both rigid and flexible sections to satisfy the demands for stability and catalysis. Because the flexibility of a protein segment is indispensable for a proteolytic attack, limited proteolysis is a superb tool to analyse both confined local fluctuations and global unfolding events in proteins. While the identification of the primary cleavage products allows the assignment of the flexible regions to the primary structure, the kinetics of proteolytic degradation enables differentiation between local fluctuations in the native protein molecule and the global unfolding process during denaturation. Modifications of the amino acid sequence in the concerned regions can tune proteolytic susceptibility and alter protein stability. In the present paper, we summarise our results on native-state and unfolded-state proteolysis of ribonuclease A (RNase A) and the effect of mutations in the detected flexible regions on the stability and unfolding of the RNase A molecule.

Selective stabilization of a labile mutant form of bovine pancreatic ribonuclease A by antibodies

The region between the amino acids 31-46 was previously identified as being first exposed during thermal unfolding of bovine pancreatic ribonuclease A (RNase). The exchange of one amino acid (Leu35toSer) in this unfolded region of RNase is shown to have a dramatic destabilizing effect (ΔTm=9 °C). Antibodies raised against a peptide corresponding to the sequence of the labile region, S32-V43, of RNase were effective in stabilizing L35S-RNase against thermal inactivation (65 °C for 2 h) and surpassed the stabilization effect of antiRNase antibodies. An 11% contribution to the stabilizing effect of antiRNase antibodies resulted from antibodies recognizing the unfolding region of the enzyme.

REACTIONS OF ACYLFORMYLKETENE S,S-AND S,N-ACETALS WITH AMINES

Abstract Acylformylketene S,S-and S,N-acetals 1 and 12 react with amines substituting one or both donor groups at the acetal carbon atom leading to 2–5, 7, 9–11, 13, 14 and 18 and also yielding azomethines 6–9, 11, 17 and 21. Obviously, the reaction at the acetal carbon proceeds by thermodynamic control and at the aldehyde carbon by kinetic control, 1,4-Dinucleophiles are appropriate building blocks for heterocycles 19, 20 and 22.

REACTIONS OF o-CHLOROSUBSTITUTED β-KETOENOLATES WITH HETEROCUMULENES

Abstract Treatment of o-chlorophenyl β-ketoenolates 1 with carbon disulfide in the presence of sodium hydride and subsequent alkylation with CH-acidic halocompounds affords the thieno[2,3-b]-4H-[1]benzothiin-4-ones 3. Reaction of 1 with phenyl isothiocyanate leads to thiophenes 6 which undergo intramolecular cyclization yielding thieno[2,3-b]quinolin-4-ones 7.