Probing the Dynamics of a Protein Hydrophobic Core at Low Temperatures
EMSL Project ID
34295
Abstract
Hydrophobic cores are found in the interior of globular proteins and are essential for the formation of the folded state. The core represents a complex dynamical medium reflecting the existence of a conformational ensemble. Our goal is to investigate dynamical features typical in hydrophobic cores by looking at model globular proteins. Our major experimental tool will be deuteron NMR spectroscopy, which is ideally suited for investigation of dynamics over a wide range of time scales. We choose chicken villin subdomain (HP36), which is one of the smallest examples of a cooperatively folding protein. Its small size makes it possible to use solid-state peptide synthesis for sample preparation. We can thus easily obtain several samples with mutations and isotopic labels of our choice.
Preliminary data on the wild-type HP36 sample with the deuteron label on methyl of Leuicine-69, located in the key position of the hydrophobic core, allowed us to generate a precise motional model that governs the behavior of this methyl group. The data were obtained in collaboration with PNNL staff members and Prof. R.L. Vold laboratory at the College of William and Mary. The main temperature range covered was
between 110K-298K.
At temperatures below 100K the methyl group undergoes a switch to a new motional regime and eventually reaches the limit when deuteron tunneling becomes important. Thus, the low temperature regime opens a new realm for dynamical studies.
Yet, low temperatures proved to be difficult due to low signal to noise. To alleviate the signal-to-noise constraints we propose to implement Quadrupolar Echo CPMG detection scheme for T1 relaxation times measurements. We will then use this technique to obtained T1 measurements for the wild-type HP36 sample, F58L mutant, and FMOC-leucine amino acid between 5-100K. The resulting relaxation data will be used to extract activation energies, tunneling temperatures, and heat capacities. The comparison among the three samples will allow for the elucidation of the dynamical features specific to the hydrophobic core of HP36.
To our knowledge this will be the first detailed study probing the dynamics of the core at this temperature regime by deuteron relaxation. The cryogenic probes developed at PNNL make a unique opportunity for this study. The results will advance our understanding of complex dynamical behavior of globular proteins.
Proposal Type: Open call, standard access, non-proprietary, general
Project Details
Project type
Exploratory Research
Start Date
2009-06-22
End Date
2010-06-27
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Comparative Dynamics of Leucine Methyl Groups in FMOC-Leucine and in a Protein Hydrophobic Core Probed by Solid-State Deuteron Nuclear Magnetic Resonance over 7-324 K Temperature Range. Liliya Vugmeyster, Dmitry Ostrovsky, Mark Moses, Joseph J. Ford, Andrew S. Lipton, Gina L. Hoatson, and Robert L. Vold , J. Phys. Chem. B. Web Publication November 15, 2010; doi: 10.1021/jp1082467
Freezing of Dynamics of a Methyl Group in a Protein Hydrophobic Core at Cryogenic Temperatures by Deuteron NMR Spectroscopy. Liliya Vugmeyster, Dmitry Ostrovsky, Joseph J. Ford, Andrew S. Lipton University of Alaska Anchorage, Anchorage, Alaska, 99508; Pacific Northwest National Laboratory, Richland, Washington, 99354 J. Am. Chem. Soc. 132 (12), 4038?4039 (2010)
Freezing of Dynamics of a Methyl Group in a Protein Hydrophobic Core at Cryogenic Temperatures by Deuteron NMR Spectroscopy.
Liliya Vugmeyster, Dmitry Ostrovsky, Joseph J. Ford, Andrew S. Lipton
University of Alaska Anchorage, Anchorage, Alaska, 99508;
Pacific Northwest National Laboratory, Richland, Washington, 99354
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