The role of aromatic phenylalanine residues in binding carotenoid to light-harvesting model and wild-type complexes.
The mode of carotenoid (Crt) binding to polypeptide and specifying its function is as yet largely unknown. Statistical analysis of major photosystems I and II suggests that aromatic residues make up a significant part of the Crt binding pockets. Phenylalanine residues ensure approximately 25%--at some carbon atoms even up to 40%--of the total contacts with Crts. By use of an alanine-leucine model transmembrane helix that replaces the native helix of the bacterial light-harvesting complex 2 (LH2) alpha-subunit, we study the effects of polypeptide residues on cofactor binding in a model sequence context. Here, it is shown that phenylalanine residues located in the close vicinity of the Crts' polyene backbone significantly contribute to the binding of the Crt to the model protein. The replacement of a phenylalanine with leucine in the model helix results in significant reduction in the complexes' Crt content. This effect is strongly enhanced by the removal of a second phenylalanine in close vicinity to the Crt, i.e., of the wild-type (WT) beta-subunit. Remarkably, the mutation of only two phenylalanine residues in the LH2 WT sequence, alpha-Phe at position -12 and beta-Phe at -8, results in the loss of nearly 50% of functional Crt. Resonance Raman spectra indicate that the Crt conformation is fundamentally altered by the absence of the phenylalanines' aromatic side chains, suggesting that they lock the Crt into a precise, well-defined configuration. Thus, binding and specific functionalisation of Crt in the model and WT light-harvesting complex is reliant on the aromatic residue phenylalanine. The use of the light-harvesting complex as a model system thus substantiates the notion that the aromatic residue phenylalanine is a key factor for the binding of Crt to transmembrane proteins.
García-Martín
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A
Department Biologie I, Botanik, LMU München, Menzingerstrasse 67, 80638 Munich, Germany.
Pazur
A
A
Wilhelm
B
B
Silber
M
M
Robert
B
B
Braun
P
P
D000595:Amino Acid Sequence; D002338:Carotenoids / Q000737:chemistry / Q000378:metabolism*; D045342:Light-Harvesting Protein Complexes / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism*; D045331:Photosystem I Protein Complex / Q000737:chemistry / Q000378:metabolism; D045332:Photosystem II Protein Complex / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D012242:Rhodobacter sphaeroides / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013059:Spectrum Analysis, Raman; D013329:Structure-Activity Relationship
D045342:Light-Harvesting Protein Complexes; D050505:Mutant Proteins; D045331:Photosystem I Protein Complex; D045332:Photosystem II Protein Complex; D002338:Carotenoids; D010649:Phenylalanine