SEQUENCING N-TERMINALLY BLOCKED PROTEINS

Project: Research project

Project Details

Description

Automated Edman degradation of proteins and peptides is of crucial
importance in providing amino acid sequence information for identifying and
sequencing DNA clones that contain the corresponding structural genes. The
complex of procedures encompassing protein/peptide sample preparation, the
sequencing chemistry itself, and PTH amino acid identification, has become
a high technology that is constantly being modified to improve sensitivity
and range of applicability, so that proteins of lower abundance and
increasingly diverse properties can be analyzed. At the present time, the
most important limitation to the technology is that approximately 80% of
proteins are blocked tot he Edman degradation by Nalpha- acylation of the
N-terminal residue. The significance of this limitation derives from the
preeminent importance of sequence information acquired from the N-terminus
of the intact protein for gene cloning and sequencing studies. Although
internal amino acid sequence information can generally be acquired from
blocked proteins, its acquisition always consumes considerably larger
amounts of protein than an N-terminal sequence analysis, and the lack of an
N-terminal sequence subsequently makes the DNA sequence data more difficult
to obtain and interpret.

This proposal seeks to establish general methods for deblocking picomole
amounts of proteins so that high sensitivity amino acid sequencing can be
used to determine the N-terminal sequence. A procedure for rendering small
amounts of protein susceptible to attach by specific deblocking enzymes
will be sought for implementation in a working protein chemistry
laboratory. The procedure will be based on covalent immobilization of the
substrate protein on a solid support that is compatible with Edman
sequencing. Although potentially suitable Nalpha - acetylation amino acid
hydrolyses are known that will deblock small peptide substrates, the
proposal is high risk because they have not been shown to work on longer
polypeptides. However, success of the project would result in a large
improvement in the sensitivity of amino acid sequence analysis of the
majority of proteins and would represent a major improvement in the
usefulness of the technology in molecular biology.
StatusNot started