Protein Engineering vol. 8 no. 1 pp. 63-70, 1995
© 1995 Oxford University Press
RESEARCH-ARTICLE |
Interactions of (Ala*Ala*Lys*Pro)n and (Lys*Lys*Ser*Pro)n with DNA. Proposed coiled-coil structure of AlgR3 and AlgP from Pseudomonas aeruginosa
1institute of Protein Research, Russian Academy of Sciences Pushchino, Moscow Region, 142292, Russia 2Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences Pushchino, Moscow Region, 142292, Russia 3School of Health Sciences, Sapporo Medical University Sapporo, Hokkaido 060, Japan 5Department of Biochemistry, Nagasaki University School of Medicine Nagasaki 852, Japan 6Department of Biology, University of Virginia Charlottesville, VA 22901, USA
4To whom correspondence should be addressed
7To whom correspondence should be addressed
The proteins, AlgR3 and AlgP, are involved in the regulation of alginate synthesis in Pseudomonas. They contain multiple repeats of Ala*Ala*Lys*Pro as do several other proteins that resemble histones. The interactions of synthesis oligopeptides composed of repeated Ala*Ala*Lys*Pro or Lys*Lys*Ser*Pro units with DNA were studied by fluorescence of the Fmoc (9-fluorenylmethyloxycarbonyl) group attached to the N-termini of the peptides. DNA quenching of the Fmoc fluorescence of the peptides was used to estimate the apparent association constants for the interaction of Fmoc(AAKP)nOH (n = 2, 4, 8, 18, 32) and of Fmoc(KKSP)nOH (n = 2, 4, 8, 16, 20, 32) with DNA. The Fmoc(AAKP)nOH peptides bind to DNA only at low ionic strength; the Fmoc(KKSP)n OH peptides interact with DNA at both low (0.05 M KCl) and high (0.2 M KCl) salt At low ionic strength an increase in the number of the repeat units causes an increase in the apparent association constant up to {small tilde}2 x 106 M–1 for both types of peptides at N 
24. The insertion of an AAKTA unit into the middle of the Fmoc(AAKP)8OH peptide increases its affinity to DNA. We propose a model of (AAKP)n and of its interaction with DNA. The repeat unit consists of a single turn of 
-helix followed by a bend necessitated by Pro. The resultant coiled-coil forms a right-handed superhelix with 10 AAKPs per repeat distance of {small tilde}33 Å. With only slight modification of the canonical parameters of this model the AAKP super helix fits into the major groove of B-form DNA with one AAKP tetramer per base pair repeat of 3.4 Å. The 
-amine nitrogen of Lys can form a polar hydrogen bond with a phosphate oxygen atom of the DNA backbone. A better fit is obtained when the model is modified to accommodate [(AAKP)5AAKTA]n as actually observed in AlgR3. We suggest that this coiled-coil represents a general motif for other protein–DNA interactions.
Keywords: alginate synthesis/DNA-peptide binding/fluorescence/Fmoc/histone Hl/proline bend/Pseudomonas/tandem repeat
Received September 10, 1994; revised September 29, 1994; accepted October 15, 1994.
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