Probing the role of Proline in the antimicrobial activity and lipopolysaccharide binding of indolicidin.
OBJECTIVE
Indolicidin (ILPWKWPWWPWRR-NH2), an antimicrobial peptide from bovine neutrophils, possesses significant antibacterial activity. An interesting feature of indolicidin is its unusually high content of Tryptophan and Proline residues. While the involvement of Tryptophan has been studied for its hemolytic and antibacterial activity, little is known about the roles played by Proline in these aspects. We herein investigate the structure and biological activities of indolicidin, where Proline at either one or more of the 3rd, 7th, 10th positions has been replaced by Alanine to better understand its structure and biological function.


METHODS
Structural aspects of Proline residues of indolicidin and its effect on antimicrobial activity were elucidated by replacing Proline residues with Alanine. Minimum inhibitory concentration (MIC) and scanning electron microscopy (SEM) experiments provide substantial evidence for the importance of Proline residues for antimicrobial activity and cell wall disintegration. Binding affinity of the peptides to Lipopolysaccharide (LPS) was investigated using fluorescence spectroscopy and dynamic light scattering (DLS) in conjunction with (31)PNMR spectroscopy and confirmed the disintegration of LPS layer.


RESULTS
Our study reveals that Proline residues are necessary for interaction of indolicidin with LPS and establishes the significance of the third and tenth Proline residues for its antimicrobial activity. We believe that the presence of so many Proline residues provides the molecule a selective advantage of adopting different conformations varying from a globular, closed conformation to an open extended conformation, and even to a wedge-shaped conformation, which account for the diverse mechanisms of action of indolicidin.
Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700054, India.
Swapna
S
Bera
Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700054, India.
Anirban
A
Ghosh
Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700054, India.
Shruti
S
Sharma
Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700054, India.
Tanmoy
T
Debnath
Central Instrument Facility, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700054, India.
Banabihari
B
Giri
Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700054, India. Electronic address: bhunia@jcbose.ac.in.
Anirban
A
Bhunia
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000737:chemistry* / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis / Q000737:chemistry* / Q000494:pharmacology*; D001665:Binding Sites; D002417:Cattle; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000254:growth & development; D008070:Lipopolysaccharides; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000378:metabolism; D011392:Proline / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000254:growth & development; D012485:Salmonella typhi / Q000187:drug effects / Q000254:growth & development; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry
D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D008070:Lipopolysaccharides; C073143:indolicidin; D014364:Tryptophan; D011392:Proline; D000409:Alanine
(31)P NMR spectroscopy; AMP; CD spectroscopy; DLS; Indolicidin; LPS; SEM