The inactivating factor of glutamine synthetase, IF7, is a "natively unfolded" protein.
Glutamine synthetase (GS) is the key enzyme responsible for the primary assimilation of ammonium in all living organisms, and it catalyses the synthesis of glutamine from glutamic acid, ATP, and ammonium. One of the recently discovered mechanisms of GS regulation involves protein-protein interactions with a small 65-residue-long protein named IF7. Here, we study the structure and stability of IF7 and its binding properties to GS, by using several biophysical techniques (fluorescence, circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopies, and gel filtration chromatography) which provide complementary structural information. The findings show that IF7 has a small amount of residual secondary structure, but lacks a well defined tertiary structure, and is not compact. Thus, all of the studies indicate that IF7 is a "natively unfolded" protein. The binding of IF7 to GS, its natural binding partner, occurs with an apparent dissociation constant of K(D) = 0.3 +/- 0.1 microM, as measured by fluorescence. We discuss the implications for the GS regulation mechanisms of IF7 being unfolded.
Muro-Pastor
M Isabel
MI
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-Consejo Superior de Investigaciones Científicas, 41092 Seville, Spain.
Barrera
Francisco N
FN
Reyes
José C
JC
Florencio
Francisco J
FJ
Neira
José L
JL
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002850:Chromatography, Gel; D002942:Circular Dichroism; D005974:Glutamate-Ammonia Ligase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D013997:Time Factors
D001426:Bacterial Proteins; C120034:IF7 protein, Synechocystis sp.; D005974:Glutamate-Ammonia Ligase