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.
- National Center for Research Resources
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