Complete 1H nuclear magnetic resonance assignments and structural characterization of a fusion protein of the alpha-amylase inhibitor tendamistat with the activation domain of the human immunodeficiency virus type 1 Tat protein.
Complete sequence-specific assignments of the 1H-NMR spectrum of a fusion protein of the alpha-amylase inhibitor tendamistat from Streptomyces tendae and the activation domain of Tat from human immunodeficiency virus type 1 (HIV-1) was obtained by homonuclear two-dimensional NMR methods. The protein behaves as expected for an ideal fusion protein: the flexible linker allows an almost completely decoupled motion of the subunits of the protein and the two subunits show almost no mutual interaction. In the tendamistat part, small structural distortions due to exchange of the carboxy-terminal leucine propagate mainly via the hydrogen bonds of the beta-sheet and the disulfide bond. The Tat part of the protein contains the seven cysteine residues of full-length Tat. The fusion protein was expressed in Streptomyces lividans and exported. During the export to the extracellular space disulfide bonds are created by the expressing cells, only one sulfhydryl group remains accessible for sulfhydryl reagents. Although a unique, dominant conformation with a specific disulfide bonding pattern exists, a significant conformational variation can be observed including cis-proline peptide bonds, which may indicate smaller populations with alternative disulfide bonding patterns.
Freund
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J
Max-Planck-Institute for Medical Research, Department of Biophysics, Heidelberg, Germany.
Vértesy
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Koller
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Wolber
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Heintz
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Kalbitzer
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D000595:Amino Acid Sequence; D004926:Escherichia coli; D015696:Gene Products, tat / Q000737:chemistry* / Q000235:genetics; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013302:Streptomyces / Q000737:chemistry; D013455:Sulfur / Q000737:chemistry; D013696:Temperature; D000516:alpha-Amylases / Q000037:antagonists & inhibitors*; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D010455:Peptides; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D013455:Sulfur; D000516:alpha-Amylases; C030526:tendamistate