TY - JOUR
T1 - The differences in the microenvironment of the two tryptophan residues of the glutamine-binding protein from Escherichia coli shed light on the binding properties and the structural dynamics of the protein
AU - D'Auria, Sabato
AU - Staiano, Maria
AU - Varriale, Antonio
AU - Gonnelli, Margherita
AU - Marabotti, Anna
AU - Rossi, Mose'
AU - Strambini, Giovanni B.
PY - 2008/5/1
Y1 - 2008/5/1
N2 - Glutamine-binding protein (GlnBP) from Escherichia coli is a monomer (26 kDa) that is responsible for the first step in the active transport of L-glutamine across the cytoplasmic membrane. GlnBP consists of two domains (termed large and small) linked by two antiparallel β-strands. The large domain is similar to the small domain but it contains two additional α-helices and three more short antiparallel β-strands. The deep cleft formed between the two domains contains the ligand-binding site. The binding of L-glutamine leads to cleft closing and a significant structural change with the formation of the so-called "closed form" structure. The protein contains two tryptophan residues (W32 and W220) and 10 tyrosine residues. We used phosphorescence spectroscopy measurements to characterize the role of the two tryptophan residues in the protein structure in the absence and the presence of glutamine. Our results pointed out that the phosphorescence of GlnBP is easily detected in fluid solutions where the emission of the two tryptophan residues is readily discriminated by the drastic difference in the phosphorescence lifetime allowing the assignments of the short lifetime to W220 and the long lifetime to W32. In addition, our results showed that the triplet lifetime of the superficial W220 is unusually short because of intramolecular quenching by the proximal Y163. On the contrary, the lifetime of W32 is several hundred milliseconds long, implicating a well-ordered, compact fold of the surrounding polypeptide. The spectroscopic data were analyzed and discussed together with a detailed inspection of the 3D structure of GlnBP.
AB - Glutamine-binding protein (GlnBP) from Escherichia coli is a monomer (26 kDa) that is responsible for the first step in the active transport of L-glutamine across the cytoplasmic membrane. GlnBP consists of two domains (termed large and small) linked by two antiparallel β-strands. The large domain is similar to the small domain but it contains two additional α-helices and three more short antiparallel β-strands. The deep cleft formed between the two domains contains the ligand-binding site. The binding of L-glutamine leads to cleft closing and a significant structural change with the formation of the so-called "closed form" structure. The protein contains two tryptophan residues (W32 and W220) and 10 tyrosine residues. We used phosphorescence spectroscopy measurements to characterize the role of the two tryptophan residues in the protein structure in the absence and the presence of glutamine. Our results pointed out that the phosphorescence of GlnBP is easily detected in fluid solutions where the emission of the two tryptophan residues is readily discriminated by the drastic difference in the phosphorescence lifetime allowing the assignments of the short lifetime to W220 and the long lifetime to W32. In addition, our results showed that the triplet lifetime of the superficial W220 is unusually short because of intramolecular quenching by the proximal Y163. On the contrary, the lifetime of W32 is several hundred milliseconds long, implicating a well-ordered, compact fold of the surrounding polypeptide. The spectroscopic data were analyzed and discussed together with a detailed inspection of the 3D structure of GlnBP.
KW - Glutamine-binding protein
KW - Phosphorescence
KW - Protein conformation
KW - Protein dynamics
KW - Tryptophan
UR - http://www.scopus.com/inward/record.url?scp=41149173805&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=41149173805&partnerID=8YFLogxK
U2 - 10.1002/prot.21748
DO - 10.1002/prot.21748
M3 - Article
C2 - 17979189
AN - SCOPUS:41149173805
VL - 71
SP - 743
EP - 750
JO - Proteins: Structure, Function and Genetics
JF - Proteins: Structure, Function and Genetics
SN - 0887-3585
IS - 2
ER -