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Reply from Blalock
282
T I B T E C H - O C T O B E R 1990 [Vol. 8]
Reply from Blalock I entirely agree with J. Slootstra and E. Roubos that 'receptor and hormone may have originated from a single DNA molecule in which one strand coded for the hormone and the other for the receptor'. In fact, our original article stated this 1. Similarly, we concur 2 with R. Brentani that co-evolution of interacting peptides coded for by comp-
lementary codons would speed up evolution. I do, however, disagree with his contention that the X-ray crystallography and site-specific mutagenesis work was misquoted. These techniques led to identification of the same sites of interaction of interleukin-2 and its receptor as would be predicted by the Molecular Recognition Theory.
New tools for protein sequence analysis Fumio Sakiyama Analysis of protein sequence is an important tool in studies of both native and recombinant proteins. Novel techniques and instrumentation w h i c h facilitate determination of protein primary structure have recently been developed. The functional role of a protein depends on its specific threedimensional structure which is determined primarily by its amino acid sequence (primary structure). Protein sequence analysis is thus an essential step towards an understanding of a protein's function. Since the amino acid sequence is dictated by the nucleotide sequence, DNA sequencing (now a rapid, straightforward technique, relatively economical in lerms of materials required) has become a popular method of obtaining information on amino acid sequence, especially for large proteins of which only small amounts are a v a i l a b l e for study. However, this approach has the drawback that it provides no information on the structure of proteins modified post-translationally. Direct sequencing of the polypeptide chain(s) is therefore a preF. Sakiyama is at the Institute for Protein Research, Osaka University, Saita, Osaka 565, Japan.
ferred option when only nanomolar amounts of highly purified protein of molecular mass of 30 kDa or less are available. Recently, new instrumentation and improved techniques have been developed I 6. We discuss those which are now being used routinely as well as those which will become of practical importance in the near future. The strategy for protein sequence analysis has remained essentially unchanged for the past 30 years, and employs a series of reactions termed the Edman degradation. I n this, phenylisothiocyanate is reacted with the terminal amino group of a protein/peptide yielding a derivative which may be cleaved from the peptide chain. The cleaved residue undergoes a rearrangement to a phenylthiohydantoin (PTH) derivative of the amino acid, easily identified by HPLC. Repetition of this sequence of reactions permits the sequencing of the complete polypeptide, starting from the N-terminal
1990, Elsevier Science Publishers Ltd (UK) 0167- 9430/90/$2.00
References 1 Bost, K. L., Smith, E. M. and Blalock, J. E. (1985) Proc. Natl Acad. Sci. USA 82, 1372-1375 2 Blalock, J. E. and Bost, K. L. (1988) Recent Prog. Horm. Res. 44, 199-222 J. E D W I N B L A L O C K
Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
end. Nowadays the majority of direct sequencing by Edman degradation is carried out in sequenators with most of the process automated.
Protein sequence analysis procedure A typical protein sequence analysis procedure consists of eight steps: (1) N-terminal analysis; (2) amino acid analysis; (3) protein fragmentation; (4) peptide fractionation; (5) Edman degradation and PTH-amino acid analysis; (6) C-terminal analysis; (7) location of the disulfide bond; and (8) determination of the mode and site of post translational modification. Step 1: N-terminal analysis This can be u s e d not only to determine the amino acid sequence of the N-terminal region but also to test the purity of the protein sample. A gas-phase or pulsed-liquid sequencer (see Glossary~ equipped with PTH-amino acid analyser can determine the 30-40 N-terminal amino acids by direct sequencing of 50-100 pmol of a protein. Under optimal running conditions, up to 10 amino acid residues may be determined from 5-10 pmol of a protein. The N-terminal sequencing of a protein which has been separated by SDS-PAGE and blotted to a polyvinylidenedifluoride (PVDF) (see Glossary) membrane is routine 7. Frequently, in the direct sequence analysis, a PTH-amino acid cannot be detected in the first cycle of Edman degradation of a protein. In most cases, this is due to the reaction being blocked by an acyl group at the N-terminus. The presence of an acetyl group at the N-terminus is a
T I B T E C H - O C T O B E R 1990 [Vol. 8]
Reply from Blalock I entirely agree with J. Slootstra and E. Roubos that 'receptor and hormone may have originated from a single DNA molecule in which one strand coded for the hormone and the other for the receptor'. In fact, our original article stated this 1. Similarly, we concur 2 with R. Brentani that co-evolution of interacting peptides coded for by comp-
lementary codons would speed up evolution. I do, however, disagree with his contention that the X-ray crystallography and site-specific mutagenesis work was misquoted. These techniques led to identification of the same sites of interaction of interleukin-2 and its receptor as would be predicted by the Molecular Recognition Theory.
New tools for protein sequence analysis Fumio Sakiyama Analysis of protein sequence is an important tool in studies of both native and recombinant proteins. Novel techniques and instrumentation w h i c h facilitate determination of protein primary structure have recently been developed. The functional role of a protein depends on its specific threedimensional structure which is determined primarily by its amino acid sequence (primary structure). Protein sequence analysis is thus an essential step towards an understanding of a protein's function. Since the amino acid sequence is dictated by the nucleotide sequence, DNA sequencing (now a rapid, straightforward technique, relatively economical in lerms of materials required) has become a popular method of obtaining information on amino acid sequence, especially for large proteins of which only small amounts are a v a i l a b l e for study. However, this approach has the drawback that it provides no information on the structure of proteins modified post-translationally. Direct sequencing of the polypeptide chain(s) is therefore a preF. Sakiyama is at the Institute for Protein Research, Osaka University, Saita, Osaka 565, Japan.
ferred option when only nanomolar amounts of highly purified protein of molecular mass of 30 kDa or less are available. Recently, new instrumentation and improved techniques have been developed I 6. We discuss those which are now being used routinely as well as those which will become of practical importance in the near future. The strategy for protein sequence analysis has remained essentially unchanged for the past 30 years, and employs a series of reactions termed the Edman degradation. I n this, phenylisothiocyanate is reacted with the terminal amino group of a protein/peptide yielding a derivative which may be cleaved from the peptide chain. The cleaved residue undergoes a rearrangement to a phenylthiohydantoin (PTH) derivative of the amino acid, easily identified by HPLC. Repetition of this sequence of reactions permits the sequencing of the complete polypeptide, starting from the N-terminal
1990, Elsevier Science Publishers Ltd (UK) 0167- 9430/90/$2.00
References 1 Bost, K. L., Smith, E. M. and Blalock, J. E. (1985) Proc. Natl Acad. Sci. USA 82, 1372-1375 2 Blalock, J. E. and Bost, K. L. (1988) Recent Prog. Horm. Res. 44, 199-222 J. E D W I N B L A L O C K
Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
end. Nowadays the majority of direct sequencing by Edman degradation is carried out in sequenators with most of the process automated.
Protein sequence analysis procedure A typical protein sequence analysis procedure consists of eight steps: (1) N-terminal analysis; (2) amino acid analysis; (3) protein fragmentation; (4) peptide fractionation; (5) Edman degradation and PTH-amino acid analysis; (6) C-terminal analysis; (7) location of the disulfide bond; and (8) determination of the mode and site of post translational modification. Step 1: N-terminal analysis This can be u s e d not only to determine the amino acid sequence of the N-terminal region but also to test the purity of the protein sample. A gas-phase or pulsed-liquid sequencer (see Glossary~ equipped with PTH-amino acid analyser can determine the 30-40 N-terminal amino acids by direct sequencing of 50-100 pmol of a protein. Under optimal running conditions, up to 10 amino acid residues may be determined from 5-10 pmol of a protein. The N-terminal sequencing of a protein which has been separated by SDS-PAGE and blotted to a polyvinylidenedifluoride (PVDF) (see Glossary) membrane is routine 7. Frequently, in the direct sequence analysis, a PTH-amino acid cannot be detected in the first cycle of Edman degradation of a protein. In most cases, this is due to the reaction being blocked by an acyl group at the N-terminus. The presence of an acetyl group at the N-terminus is a