Nuclear Magnetic Resonance Diffusion on Nucleotide Binding with HSA
Primary Faculty Mentor’s Name
Zhiyan Song
Proposal Track
Student
Session Format
Poster
Abstract
The pulsed-field gradient (PFG) 31P NMR diffusion spectra were measured for guanosine or adenosine nucleotide samples in absence or presence of human serum albumin (HSA). Human serum albumin is the most abundant protein in human blood plasma. It is produced in the liver. Albumin constitutes about half of the blood serum protein. It is soluble and monomeric. The NMR samples (including GTP, GDP, GMP, ATP, ADP or AMP, and in the presence or absence of HSA) were fully dissolved in 1mL of D2O for the purpose magnetic field lock. The nucleotide concentration was 10 mM, and HSA concentration was 1mM. The sample pH (=6.99) was calibrated with either NaOH or HCl. The prepared samples were incubated for a few hours prior to NMR acquisition to ensure the equilibrium was reached. 31P NMR experiment was carried out using a Jeol Ecx-300 spectrometer. A standard stimulated spin-echo (SSE) pulse sequence was used with 256 scans; 3 s repetition delay; 10.75 μs of π/2 pulse-length; 200 ms of diffusion time (Δ); 4 ms of gradient-pulse duration ( δ); and gradient-pulse strength (G) incremented from 0.003 to 0.283 Tesla/m. We found that the interaction of nucleotides with HSA has no significant effects on the 31P chemical shift values and line-shapes of nucleotide spectra, but it can significantly decrease the nucleotide diffusions. The observed nucleotide diffusion constants of the samples were derived from the diffusion spectra, and were used to characterize the weak binding interaction between nucleotides and HSA. By comparing the data, the relative binding affinities of different nucleotides on HSA were established in the order of GMP > GDP > GTP for guanosine nucleotides, and AMP > ADP > ATP for adenosine nucleotides, respectively. These results are important for elucidating the weak affinity interaction of nucleotides with serum albumin and its possible impact. This method can be applicable for investigating other nucleotide- protein interactions.
Keywords
Pulsed-field gradient, Guanosine, Adenosine, HSA, NMR, Nucleotides, Diffusion, Spectra, Weak affinity, Spectrometer
Award Consideration
1
Location
Concourse/Atrium
Presentation Year
2014
Start Date
11-15-2014 2:55 PM
End Date
11-15-2014 4:10 PM
Publication Type and Release Option
Presentation (Open Access)
Recommended Citation
Taylor, Chantia K., "Nuclear Magnetic Resonance Diffusion on Nucleotide Binding with HSA" (2014). Georgia Undergraduate Research Conference (2014-2015). 102.
https://digitalcommons.georgiasouthern.edu/gurc/2014/2014/102
Nuclear Magnetic Resonance Diffusion on Nucleotide Binding with HSA
Concourse/Atrium
The pulsed-field gradient (PFG) 31P NMR diffusion spectra were measured for guanosine or adenosine nucleotide samples in absence or presence of human serum albumin (HSA). Human serum albumin is the most abundant protein in human blood plasma. It is produced in the liver. Albumin constitutes about half of the blood serum protein. It is soluble and monomeric. The NMR samples (including GTP, GDP, GMP, ATP, ADP or AMP, and in the presence or absence of HSA) were fully dissolved in 1mL of D2O for the purpose magnetic field lock. The nucleotide concentration was 10 mM, and HSA concentration was 1mM. The sample pH (=6.99) was calibrated with either NaOH or HCl. The prepared samples were incubated for a few hours prior to NMR acquisition to ensure the equilibrium was reached. 31P NMR experiment was carried out using a Jeol Ecx-300 spectrometer. A standard stimulated spin-echo (SSE) pulse sequence was used with 256 scans; 3 s repetition delay; 10.75 μs of π/2 pulse-length; 200 ms of diffusion time (Δ); 4 ms of gradient-pulse duration ( δ); and gradient-pulse strength (G) incremented from 0.003 to 0.283 Tesla/m. We found that the interaction of nucleotides with HSA has no significant effects on the 31P chemical shift values and line-shapes of nucleotide spectra, but it can significantly decrease the nucleotide diffusions. The observed nucleotide diffusion constants of the samples were derived from the diffusion spectra, and were used to characterize the weak binding interaction between nucleotides and HSA. By comparing the data, the relative binding affinities of different nucleotides on HSA were established in the order of GMP > GDP > GTP for guanosine nucleotides, and AMP > ADP > ATP for adenosine nucleotides, respectively. These results are important for elucidating the weak affinity interaction of nucleotides with serum albumin and its possible impact. This method can be applicable for investigating other nucleotide- protein interactions.
