Dynamics of lactose permease of Escherichia coli determined by site-directed chemical labeling and fluorescence spectroscopy
Φόρτωση...
Ημερομηνία
Συγγραφείς
Wu, J.
Frillingos, S.
Kaback, H. R.
Τίτλος Εφημερίδας
Περιοδικό ISSN
Τίτλος τόμου
Εκδότης
Περίληψη
Τύπος
Είδος δημοσίευσης σε συνέδριο
Είδος περιοδικού
peer-reviewed
Είδος εκπαιδευτικού υλικού
Όνομα συνεδρίου
Όνομα περιοδικού
Biochemistry
Όνομα βιβλίου
Σειρά βιβλίου
Έκδοση βιβλίου
Συμπληρωματικός/δευτερεύων τίτλος
Περιγραφή
Mutants with a single Cys residue in place of Phe27, Pro28, Phe29, Phe30, or Pro31 at the periplasmic end of putative transmembrane helix I were used to study the interaction of lactose permease with ligand by site-directed chemical modification or fluorescence spectroscopy. With permease embedded in the native membrane, mutant Phe27-->Cys or Phe28-->Cys is readily labeled with [14C]-N-ethylmaleimide (NEM), while mutant Phe29-->Cys, Phe30-->Cys, or Phe31-->Cys reacts less effectively. beta,D-Galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) has little or no effect on the reactivity of Phe27-->Cys, Phe29-->Cys, or Phe30-->Cys permease. Remarkably, however, Pro31-->Cys permease which is essentially unreactive in the absence of ligand becomes highly reactive in the presence of TDG. Ligand also enhances the NEM reactivity of the mutant with Cys in place of Pro28 which is presumably on the same face of helix I as position 31. The five single-Cys mutants which also contain a biotin acceptor domain in the middle cytoplasmic loop were purified by monomeric avidin-affinity chromatography in dodecyl beta,D-maltoside and subjected to site-directed fluorescence spectroscopy. Mutants Phe27-->Cys, Phe29-->Cys, and Phe30-->Cys react rapidly with 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS), and reactivity is not altered in the presence of TDG. In striking contrast, mutants Pro28-->Cys and Pro31-->Cys react extremely slowly with MIANS in the absent of ligand, and TDG dramatically enhances reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)
Περιγραφή
Λέξεις-κλειδιά
Amino Acid Sequence, Anilino Naphthalenesulfonates, Carbohydrate Sequence, Cysteine, Escherichia coli/*enzymology/genetics, *Escherichia coli Proteins, Ethylmaleimide/metabolism, Fluorescent Dyes, Kinetics, Membrane Transport Proteins/biosynthesis/*chemistry, Molecular Sequence Data, *Monosaccharide Transport Proteins, Mutagenesis, Site-Directed, *Protein Conformation, *Protein Structure, Secondary, Pyrenes, Recombinant Proteins/biosynthesis/chemistry, Spectrometry, Fluorescence, Sulfhydryl Reagents, *Symporters, Thiogalactosides
Θεματική κατηγορία
Παραπομπή
Σύνδεσμος
http://www.ncbi.nlm.nih.gov/pubmed/7599118
http://pubs.acs.org/doi/pdf/10.1021/bi00026a007
http://pubs.acs.org/doi/pdf/10.1021/bi00026a007
Γλώσσα
en
Εκδίδον τμήμα/τομέας
Όνομα επιβλέποντος
Εξεταστική επιτροπή
Γενική Περιγραφή / Σχόλια
Ίδρυμα και Σχολή/Τμήμα του υποβάλλοντος
Πανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Τμήμα Ιατρικής