Related Experiment Videos
Structure and dynamics of bacteriophage IKe major coat protein in MPG micelles by solution NMR
K A Williams1, N A Farrow, C M Deber
1Division of Biochemistry Research, Hospital for Sick Children, Toronto, Ontario, Canada.
Abstract:
The structure and dynamics of the 53-residue filamentous bacteriophage IKe major coat protein in fully protonated myristoyllysophosphatidylglycerol (MPG) micelles were characterized using multinuclear solution NMR spectroscopy. Detergent-solubilized coat protein [sequence: see text] mimics the membrane-bound "assembly intermediate" form of the coat protein which occurs during part of the phage life cycle. NMR studies of the IKe coat protein show that the coat protein is largely alpha-helical, exhibiting a long amphipathic surface. helix (Asn 4 to Ser 26) and a shorter "micelle-spanning" C-terminal helix which begins at TRP 29 and continues at least to Phe 48. Pro 30 likely occurs in the first turn of the C-terminal helix, where it is ideally situated given the hydrogen bonding and steric restrictions imposed by this residue. The similarity of 15N relaxation values (T1, T2, and NOE and 500 MHz and T2 at 600 MHz) among much of the N-terminal helix and all of the TM helix indicates that the N-terminal helix is as closely associated with the micelle as the TM helix. The description of the protein in the micelle is supported by the observation of NOEs between lysolipid protons and protein amide protons between asn 8 and Ser 50. The N-terminal and TM helices exhibit substantial mobility on the microsecond to second time scale, which likely reflects changes in the orientation between the two helices. The overall findings serve to clarify the role of individual residues in the context of a TM alpha-helix and provide an understanding of the secondary structure, dynamics, and aqueous and micellar environments of the coat protein.
Insights
The filamentous bacteriophage IKe coat protein is largely alpha-helical in MPG micelles, mimicking its membrane-bound state. NMR studies reveal dynamic N-terminal and C-terminal helices associated with the micelle.
Area of Science:
- Structural Biology
- Biophysics
- Molecular Microbiology
Background:
- The filamentous bacteriophage IKe major coat protein plays a crucial role in the phage life cycle.
- Understanding its structure and dynamics is key to elucidating viral assembly mechanisms.
- The protein exists in a membrane-bound "assembly intermediate" form during infection.
Purpose of the Study:
- To characterize the structure and dynamics of the IKe major coat protein within myristoyllysophosphatidylglycerol (MPG) micelles.
- To investigate the protein's secondary structure, dynamics, and micellar environment.
- To clarify the role of individual residues in a transmembrane alpha-helix context.
Main Methods:
- Multinuclear solution Nuclear Magnetic Resonance (NMR) spectroscopy was employed.
- The IKe coat protein was studied in fully protonated MPG micelles.
- 15N relaxation measurements (T1, T2, NOE) and Nuclear Overhauser Effect (NOE) experiments were performed.
Main Results:
- The IKe coat protein is predominantly alpha-helical, featuring a long amphipathic N-terminal helix and a shorter C-terminal helix spanning the micelle.
- Both N-terminal and C-terminal helices show significant association with the micelle, indicated by relaxation data and NOEs with lysolipid protons.
- Substantial microsecond-to-second timescale mobility was observed between the N-terminal and C-terminal helices.
Conclusions:
- The detergent-solubilized coat protein structure in MPG micelles effectively mimics the membrane-bound assembly intermediate.
- The findings provide insights into the secondary structure, dynamics, and environmental interactions of the IKe coat protein.
- The study clarifies the functional roles of specific residues within the transmembrane alpha-helix structure.