TY - JOUR
T1 - Distance determination in proteins using designed metal ion binding sites and site-directed spin labeling
T2 - Application to the lactose permease of Escherichia coli
AU - Voss, John
AU - Hubbell, Wayne L.
AU - Kaback, H. Ronald
PY - 1995/12/19
Y1 - 1995/12/19
N2 - As shown in the accompanying paper, the magnetic dipolar interaction between site-directed metalnitroxide pairs can be exploited to measure distances in T4 lysozyme, a protein of known structure. To evaluate this potentially powerful method for general use, particularly with membrane proteins that are difficult to crystallize, both a paramagnetic metal ion binding site and a nitroxide side chain were introduced at selected positions in the lactose permease of Escherichia coli, a paradigm for polytopic membrane proteins. Thus, three individual cysteine residues were introduced into putative helix IV of a lactose permease mutant devoid of native cysteine residues containing a high-affinity divalent metal ion binding site in the form of six contiguous histidine residues in the periplasmic loop between helices III and IV. In addition, the construct contained a biotin acceptor domain in the middle cytoplasmic loop to facilitate purification. After purification and spin labeling, electron paramagnetic resonance spectra were obtained with the purified proteins in the absence and presence of Cu(II). The results demonstrate that positions 103, 111, and 121 are 8, 14, and >23 Å from the metal binding site. These data are consistent with an α-helical conformation of transmembrane domain IV of the permease. Application of the technique to determine helix packing in lactose permease is discussed.
AB - As shown in the accompanying paper, the magnetic dipolar interaction between site-directed metalnitroxide pairs can be exploited to measure distances in T4 lysozyme, a protein of known structure. To evaluate this potentially powerful method for general use, particularly with membrane proteins that are difficult to crystallize, both a paramagnetic metal ion binding site and a nitroxide side chain were introduced at selected positions in the lactose permease of Escherichia coli, a paradigm for polytopic membrane proteins. Thus, three individual cysteine residues were introduced into putative helix IV of a lactose permease mutant devoid of native cysteine residues containing a high-affinity divalent metal ion binding site in the form of six contiguous histidine residues in the periplasmic loop between helices III and IV. In addition, the construct contained a biotin acceptor domain in the middle cytoplasmic loop to facilitate purification. After purification and spin labeling, electron paramagnetic resonance spectra were obtained with the purified proteins in the absence and presence of Cu(II). The results demonstrate that positions 103, 111, and 121 are 8, 14, and >23 Å from the metal binding site. These data are consistent with an α-helical conformation of transmembrane domain IV of the permease. Application of the technique to determine helix packing in lactose permease is discussed.
KW - electron paramagnetic resonance
KW - helix packing
KW - transmembrane helices
UR - http://www.scopus.com/inward/record.url?scp=0029614376&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0029614376&partnerID=8YFLogxK
U2 - 10.1073/pnas.92.26.12300
DO - 10.1073/pnas.92.26.12300
M3 - Article
C2 - 8618889
AN - SCOPUS:0029614376
VL - 92
SP - 12300
EP - 12303
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 26
ER -