Detection of Specific Zinc Finger Peptide Complexes with Matrix-Assisted Laser Desorption
145
of multiple Zn 2 +-adducts to p55F1-d(TTGTT) is observed. We assume that these
are nonspecific adducts of p55Fl with the oligodeoxynucleotide, in which the oligodeoxynucleotide binds multiple Zn 2 + ions through its phospho diester backbone. This hypothesis is confirmed by the observation of multiple Zn 2 + -adducts
to the oligodeoxynucleotide alone and to its dimer.
To further confirm the specificity of the triple complex, another peptide,
luteinizing hormone releasing hormone, LHRH, was added to the sample (Fig.
9.4D). LHRH contains a histidine residue in its sequence and thus potentially
binds to Zn 2 + ions. It also contains arginine with a positively charged side chain,
which may bind through electrostatic forces to the oligodeoxynucleotide backbone. Both interactions would be nonspecific. The spectrum shows that neither
a Zn 2 + -adduct to this peptide nor a triple complex with Zn 2 + and d(TTGTT) is
detected. The corresponding complex signals of p55Fl are as intense as in the
absence of LHRH. This experiment supports a specific complex formation of
Zn2+ with p55F1 and d(TTGTT) which can be followed with MALDI.
An experiment demonstrating the effect of a chemical modification of the zinc
finger peptide on its complexation to the oligodeoxynucleotide was also performed. Since the binding of p55F1 to oligodeoxynucleotides is important for
HIV replication, such a modification mimics the action of an antiviral agent. For
this purpose, Cu 2 + was added to a sample containing peptide, Zn 2 + and the oligodeoxynucleotide. It is known from solution experiments that this metal ion oxidizes the peptide's thiol functions on the zinc complexing cysteine residues to an
intramolecular disulfide bond by simultaneous reduction of Cu 2 + to Cu+. The
modified p55F1 forms complexes neither with Zn 2 + nor with oligodeoxynucleotides. This is also reflected in the MALDI spectrum in Fig. 9.4E: if Cu 2 + is added to
the sample, the cysteine residues are oxidized and the signal of the triple complex
disappears. A strong p55Fl dimer is detected instead, because peptide oxidation
can also lead to intermolecular disulfide bonds involving the third cysteine thiol.
This experiment therefore indicates that MALDI MS may be a potential method
for rapidly screening antiviral HIV agents.
Besides the complexation of Zn-p55Fl with d(TTGTT), other sequences, like
d(TTATT) and d(ACGCC) were also investigated. Our MALDI experiments with
d(TTATT) instead of d(TTGTT) support the findings ofLam et al. 1994 and Gorelick et al. 1993 who report that Zn-p55F1 is not sequence-specific in solution. We
found that the complex of d(TTATT) with Zn-p55F1 gave signals of about the
same intensity as with d(TTGTT). MALDI experiments with oligodeoxynucleotide sequences lacking thymidine are, unfortunately, not very informative. If
d(ACGCC) was used as the binding partner hardly any MALDI signal could be
seen at all, although the corresponding triple complex should be stable in solution (South and Summers 1993). It is common knowledge that oligodeoxynucleotides that do not contain thymi dines are much less stable as ions and thus much
more difficult to detect by MALDI MS. Also, they may crystallize less favorably
with the matrix, using the same sample preparation, than those containing
mostly thymidines (Schneider and Chait 1993).
The experiments probing the sequence specificity of p55F1 reveal a current
limitation of MALDI: a "MALDI window" in which the target complex is
observed has to be found. The MALDI window is given by the experimental sam-
145
of multiple Zn 2 +-adducts to p55F1-d(TTGTT) is observed. We assume that these
are nonspecific adducts of p55Fl with the oligodeoxynucleotide, in which the oligodeoxynucleotide binds multiple Zn 2 + ions through its phospho diester backbone. This hypothesis is confirmed by the observation of multiple Zn 2 + -adducts
to the oligodeoxynucleotide alone and to its dimer.
To further confirm the specificity of the triple complex, another peptide,
luteinizing hormone releasing hormone, LHRH, was added to the sample (Fig.
9.4D). LHRH contains a histidine residue in its sequence and thus potentially
binds to Zn 2 + ions. It also contains arginine with a positively charged side chain,
which may bind through electrostatic forces to the oligodeoxynucleotide backbone. Both interactions would be nonspecific. The spectrum shows that neither
a Zn 2 + -adduct to this peptide nor a triple complex with Zn 2 + and d(TTGTT) is
detected. The corresponding complex signals of p55Fl are as intense as in the
absence of LHRH. This experiment supports a specific complex formation of
Zn2+ with p55F1 and d(TTGTT) which can be followed with MALDI.
An experiment demonstrating the effect of a chemical modification of the zinc
finger peptide on its complexation to the oligodeoxynucleotide was also performed. Since the binding of p55F1 to oligodeoxynucleotides is important for
HIV replication, such a modification mimics the action of an antiviral agent. For
this purpose, Cu 2 + was added to a sample containing peptide, Zn 2 + and the oligodeoxynucleotide. It is known from solution experiments that this metal ion oxidizes the peptide's thiol functions on the zinc complexing cysteine residues to an
intramolecular disulfide bond by simultaneous reduction of Cu 2 + to Cu+. The
modified p55F1 forms complexes neither with Zn 2 + nor with oligodeoxynucleotides. This is also reflected in the MALDI spectrum in Fig. 9.4E: if Cu 2 + is added to
the sample, the cysteine residues are oxidized and the signal of the triple complex
disappears. A strong p55Fl dimer is detected instead, because peptide oxidation
can also lead to intermolecular disulfide bonds involving the third cysteine thiol.
This experiment therefore indicates that MALDI MS may be a potential method
for rapidly screening antiviral HIV agents.
Besides the complexation of Zn-p55Fl with d(TTGTT), other sequences, like
d(TTATT) and d(ACGCC) were also investigated. Our MALDI experiments with
d(TTATT) instead of d(TTGTT) support the findings ofLam et al. 1994 and Gorelick et al. 1993 who report that Zn-p55F1 is not sequence-specific in solution. We
found that the complex of d(TTATT) with Zn-p55F1 gave signals of about the
same intensity as with d(TTGTT). MALDI experiments with oligodeoxynucleotide sequences lacking thymidine are, unfortunately, not very informative. If
d(ACGCC) was used as the binding partner hardly any MALDI signal could be
seen at all, although the corresponding triple complex should be stable in solution (South and Summers 1993). It is common knowledge that oligodeoxynucleotides that do not contain thymi dines are much less stable as ions and thus much
more difficult to detect by MALDI MS. Also, they may crystallize less favorably
with the matrix, using the same sample preparation, than those containing
mostly thymidines (Schneider and Chait 1993).
The experiments probing the sequence specificity of p55F1 reveal a current
limitation of MALDI: a "MALDI window" in which the target complex is
observed has to be found. The MALDI window is given by the experimental sam-