Limited proteolysis

Multi-domain proteins may be difficult to crystallize. Often, only one domain is of interest for drug design. The full-length protein can be truncated and smaller regions or designed domains obtained which may be more amenable to crystallization. This is achieved by Limited Proteolysis, where special enzymes are used to break up the full-length protein into smaller pieces. These pieces are then characterized by Mass Spectrometry (to identify their molecular mass) and N-terminal sequencing (to locate them into the full-length protein sequence).

Thermofluor

Target proteins used for crystallization can be studied by Thermofluor, or Thermofluorescence Shift Assay. In this method, the protein is incubated with a dye that only binds hydrophobic protein regions, where it becomes highly fluorescent. The protein is gradually heated, in order to slowly unfold, exposing these hydrophobic patches. The fluorescence signal is then used to determine the protein melting point (Tm). The value of Tm is influenced not only by the nature of the protein itself but also by its chemical environment (e.g., pH, salt nature and concentration, presence of natural or synthetic ligands, etc.), and it is known empirically that the more stable is a given protein (higher Tm) the more likely it is to crystallize.

Mass spectrometry

This Mass spectrometry laboratory provides services of molecular mass determination by mass spectrometry using MALDI and electrospray ionization, LC-MS and LC-MS/MS protein characterization as well as identification and characterization, quantification and detection of small compounds. CHNS elemental analysis of organic compounds is another service of this laboratory. iBET acess through ITQB which is one of the institutions in the National Network for Mass Spectrometrywhich .

For further information please visit Mass spectrometry page

N-terminal Analysis

The N-terminal analysis of the peptides obtained from limited proteolysis is carried out by the Protein Sequencing Service of the Analytical Services Unit, which is jointly operated by ITQB and IBET.

For further information please visit Protein sequencing page