Genetic selection for and molecular dynamic modeling of a protein transmembrane domain multimerization motif from a random Escherichia coli genomic library.
In order to identify new transmembrane helix packing motifs in naturally occurring proteins, we have selected transmembrane domains from a library of random Escherichia coli genomic DNA fragments and screened them for homomultimerization via their abilities to dimerize the bacteriophage lambda cI repressor DNA-binding domain. Sequences were isolated using a modified lambda cI headpiece dimerization assay system, which was shown previously to measure transmembrane helix-helix association in the E. coli inner membrane. Screening resulted in the identification of several novel sequences that appear to mediate helix-helix interactions. One sequence, representing the predicted sixth transmembrane domain (TM6) of the E. coli protein YjiO, was chosen for further analysis. Using site-directed mutagenesis and molecular dynamics, a small set of models for YjiO TM6 multimerization interface interactions were generated. This work demonstrates the utility of combining in vivo genetic tools with computational systems for understanding membrane protein structure and assembly.
Leeds
J A
JA
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.
Boyd
D
D
Huber
D R
DR
Sonoda
G K
GK
Luu
H T
HT
Engelman
D M
DM
Beckwith
J
J
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D010582:Bacteriophage lambda / Q000276:immunology / Q000502:physiology; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D004268:DNA-Binding Proteins; D019281:Dimerization; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors; D015698:Genomic Library; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins