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Structural information on a membrane transport protein from nuclear magnetic resonance spectroscopy using
P J Spooner1, L M Veenhoff, A Watts
1Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, United Kingdom.
Biochemistry
|July 28, 1999
Summary
Researchers studied the lactose transport protein (LacS) from Streptococcus thermophilus. They found that interhelix loop 10-11 is close to the substrate binding site, impacting lactose transport.
Area of Science:
- Biochemistry
- Structural Biology
- Membrane Protein Research
Background:
- Streptococcus thermophilus lactose permease (LacS) is a key transporter protein.
- Understanding the structural dynamics of LacS is crucial for elucidating its transport mechanism.
Purpose of the Study:
- To investigate the structural role of interhelix loop 10-11 in the LacS lactose transporter.
- To determine the proximity of loop 10-11 to the substrate binding site.
Main Methods:
- Overexpression of a cysteine-mutated LacS protein (LacS K373C) in native membranes.
- Utilizing 13C-labeled substrate with solid-state NMR spectroscopy for binding studies.
- Employing electron spin resonance (ESR) and saturation-transfer ESR with nitroxide spin-labeling at K373C.
Main Results:
- NMR successfully detected substrate binding to LacS K373C in native membranes.
- Spin-labeling at K373C indicated immobility of loop 10-11 on ESR timescales.
- The spin-label obscured NMR signals, suggesting proximity (<15 Å) to the substrate binding site.
Conclusions:
- Interhelix loop 10-11 is located near the substrate binding site of LacS.
- This loop likely plays a role in the translocation process by interacting with transmembrane segments.