Control of lysine reactivity in four-helix bundle proteins by site-selective pKa depression: expanding the versatility of proteins by postsynthetic functionalization.
Five 42-residue polypeptides have been designed to fold into hairpin helix-loop-helix motifs that dimerize to form four-helix bundles, and to serve as protein scaffolds for the elucidation at the molecular level of the principles that control and fine-tune lysine and ornithine reactivities in a protein context. Site-selective control of Lys and Orn reactivity provides a mechanism for addressing directly individual residues and is a prerequisite for the site-selective functionalization of folded proteins. Several lysine and one ornithine residues were introduced on the surface and in the hydrophobic core of the folded motif. The reactivity of each residue was determined by measuring the degree of acylation of the trypsin cleaved fragments by HPLC and mass spectrometry. The most reactive residues were Orn34 and Lys19, both of which were located in d positions in the heptad repeat, and therefore in hydrophobic environments. Upon reaction of the helix-loop-helix dimer KA-I with one equivalent of mono-p-nitrophenyl fumarate, Orn34 was acylated approximately three times more efficiently than Lys19, whereas Lys10 (b position), Lys15 (g position), and Lys33 (c position) remained unmodified. In the sequence KA-I-A(15) Lys15 was replaced by an alanine residue and the selectivity of Orn34 over Lys19 increased to approximately a factor of six, probably because Lys15 had the capacity to reduce the pK(a) value of Lys19 and 85 % of site-selectively monoacylated product was obtained. The pH dependence of the acylation reaction was determined and showed that the pK(a) of the reactive residues were 9.3, more than a pK(a) unit below the magnitude of the corresponding residue in a solvent exposed position. Introducing Lys and Orn residues into a or d positions of the heptad repeat therefore serves as a mechanism of depressing their pK(a) to increase their reactivity site selectively. Extensive NMR and CD spectroscopic analyses showed that the sequences fold according to prediction.
Andersson
Linda K
LK
Department of Chemistry, Göteborg University, Sweden.
Caspersson
Maud
M
Baltzer
Lars
L
D000215:Acylation; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D018257:Helix-Loop-Helix Motifs; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008239:Lysine / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry*
D010455:Peptides; D011506:Proteins; D008239:Lysine