ABSTRACT
Allostery in the binding of peptides to DNA has been studied by quantitative DNase I footprinting using four newly designed peptides containing the XP(Hyp)RK motif and N-methylpyrrole (Py) moieties. Apparent binding constants in the micromolar range as well as Hill coefficients were determined for each peptide. The results, together with previous studies on five other peptides support the proposal that interaction network cooperativity is highly preferred in DNA-peptide interactions that involve multiple recognition sites. It is envisaged that interstrand bidentate interactions participate in the relay of conformational changes between recognition sites on the complementary strands. Models for interpreting DNA allostery based upon interaction networks are outlined. Circular dichroism experiments involving the titration of peptides against a short oligonucleotide duplex indicate that some of these peptides bind in a dimeric manner to DNA via the minor groove, inducing characteristic conformational changes. These insights should prompt the design of new DNA-binding peptides for investigating allosteric interactions between peptides and DNA, as well as novel interaction networks, and ultimately may shed light upon the fundamental chemical rules that govern allostery in more complex biological process such as DNA-protein interaction networks.
Subject(s)
DNA/chemistry , Peptides/chemistry , Autoradiography , Circular Dichroism , DNA Footprinting , Deoxyribonuclease I/chemistry , Ligands , Oligonucleotides/chemistry , Protein Binding , Protein Conformation , Reverse Transcriptase Polymerase Chain Reaction , Structure-Activity RelationshipABSTRACT
Quantitative DNase I footprinting shows that three designed peptides containing N-methylpyrrole (Py) moieties display different types of network-based allosteric communication in binding to DNA: circuit type, incomplete-circuit type, and non-circuit type characterized by interstrand bidentate interactions. Positive cooperative binding of all three peptides to individual DNA binding sites is commonly observed. CD spectral characterization of the interaction between peptides and model undecanucleotide duplexes is consistent with the footprinting results and supports the allosteric model. This study provides insights relating to the interaction network nature of allostery in complex DNA-small molecule interactions.
Subject(s)
DNA Footprinting , DNA/metabolism , Deoxyribonuclease I/metabolism , Peptides/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites , Circular Dichroism , DNA/chemistry , Peptides/chemistryABSTRACT
A novel DNA footprinting method employing strong semiquinone radical species generated from a dipeptide-hydroquinone conjugate is described.
Subject(s)
Benzoquinones/chemistry , DNA Footprinting/methods , DNA/chemistry , Deoxyribonuclease I/chemistry , Dipeptides/chemistry , Binding Sites , Electrophoresis, Polyacrylamide Gel , Molecular StructureABSTRACT
Three peptide amides, HPRK(Py)(4)HPRK-NH(2) (PyH-12), HPRK(Py)(3)HPRK-NH(2) (PyH-11) and HPRK(Py)(2)HPRK-NH(2) (PyH-10), incorporating two HPRK motifs and various 4-amino-1-methylpyrrole-2-carboxylic acid residues (Py) were synthesized by solid-phase peptide methodology. The binding of these three peptides to a 5'-32P-labeled 158-mer DNA duplex (Watson fragment) and to a 5'-32P-labeled 135-mer DNA duplex (complementary Crick fragment) was investigated by quantitative DNase I footprinting. On the 158-mer Watson strand, the most distinctive DNase I blockages seen with all three peptides occur around positions 105-112 and 76-79, corresponding to the sequences 5'-GAGAAAAT-3' and 5'-CGGT-3', respectively. However, on the complementary Crick strand, only PyH-12 strongly discriminates the 5'-TTT-3' site around positions 108-110 whereas both PyH-11 and PyH-10 have moderate binding around positions 102-112 comprising the sequence 5'-ATTTTCTCCTT-3'. Possible bidentate and single interactions of the side-chain functions and alpha-amino protons of the peptides with DNA bases are discussed.
Subject(s)
Amides/metabolism , Base Sequence , Peptides/metabolism , Protein Serine-Threonine Kinases/metabolism , Amino Acid Motifs , Bacterial Proteins , Binding Sites/physiology , DNA Footprinting/methods , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/genetics , Protein Serine-Threonine Kinases/chemical synthesis , Protein Serine-Threonine Kinases/geneticsABSTRACT
Two dodecapeptide amines: (WPRK)(3)NH(2)[WR-12] and (YPRK)(3)NH(2)[YR-12], and a 30-mer polypeptide amide (SP-30) were synthesized by solid-phase peptide methodology. DNase I footprinting studies on a 117-mer DNA showed that WR-12 and YR-12 bind selectively to DNA sequences in a manner similar to SP-30 which has a repeating SPK(R)K sequence. The most distinctive blockages seen with all three peptides occur at positions 26-30, 21-24 and 38-45 around sequences 5'-GAATT-3', 5'-TAAT-3' and 5'-AAAACGAC-3', respectively. However, it appears that YR-12 is better able to extend its recognition site to include CG pairs than is SP-30. At low concentrations YR-12 was able to induce enhanced rates of DNase I cleavage at regions surrounding some of its binding sites. To obtain further quantitative data supplementary to the footprinting work, equilibrium binding experiments were performed in which the binding of the two peptides to six decanucleotide duplexes was compared. Scatchard analyses indicated that WR-12 may be more selective for oligomers containing runs of consecutive purines or pyrimidines. On the other hand, YR-12 binds better to d(CTTAGACGTC)- d(GACGTCTAAG) than to the other oligomer duplexes, denoting selectivity for evenly distributed C/G and A/T sequences.
