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Characterization of the effect of naturally occurring mutations on the chaperone function of the human HSPA1A.
Peter Nguyen, Andrei Bilog, and Nikolas Nikolaidis
Department of Biological Science, Center for Applied Biotechnology Studies, and Center for Computational and Applied Mathematics, California State University Fullerton, Fullerton, CA
 Background
 Results
Seventy-kDa heat shock proteins (Hsp70s) are molecular chaperones involved in protein folding, synthesis, and transportation across membranes.
I480N
ADP
ATP
peptide
p= 0.001
F592S
Substrate-binding Domain
p= 0.049
p= 0.004
Figure 1: Classical cell stress response pathway mediated by Hsp70s.
p= 0.04
p= 0.003
p= 0.001
Kd (M)
p= 0.003
ATPase Domain
I74T
S16Y
p= 0.001
Hsp70s function by binding and releasing unfolded or misfolded polypeptides in an ATP depending manner.
dS (J/mol*K)
Figure 4: Three-dimensional representation of the HSPA1A protein molecule showing the position of the mutations. The ATPase domain (blue) and substrate-binding domain (green) are connected by the hydrophobic linker (red). Inlets show a magnification of the WT amino acid residues (yellow) and the mutations (magenta).
Figure 2: The Hsp70 has dual characteristics - intrinsic function as an ATPase and also interaction with co-chaperones such as the J protein as well as the nucleotide exchange factors.
p= 0.01 A B C
HSPA1A is the major heat-inducible Hsp70 in humans and is a key player in several signaling pathways that regulate protein homeostasis, cell survival. This protein has been associated with a variety of human conditions including breast and ovarian cancer, atherosclerosis, and Alzheimer’s disease. Therefore, it is important to determine functional changes of this protein as they may be related with several conditions and diseases.
dH (kJ/mol) WT
p= 0.004
In humans, a major source of genetic variation results from single nucleotide polymorphisms (SNPs); they may or may not alter the encoded amino acid.
Figure 6: Statistical analysis of WT compared to the mutant HSPA1A variants. Isothermal titration calorimetry revealed that the I480N mutant differs significantly in its affinity for ADP, ATP, and peptide substrate. This mutant also displayed significant different reaction entropy as compared to the WT HSPA1A (N=4; bars= S.D.; p values are the results of a student’s t-test). The S16Y mutant differs significantly in its affinity to both ADP and ATP. This mutant also displayed different reaction entropy and enthalpy from WT HSPA1A. N=3; bars= S.D.; p-values are the results of a student’s t-test.
S16Y
Figure 3: Single nucleotide polymorphisms (SNPs) are differences in one nucleotide base in a DNA sequence.
 Conclusions
 Purpose
I74T
Particular mutations significantly alter the binding affinity to both nucleotide and protein substrates.
Some mutations resulted in changes in the entropy and enthalpy of the reactions, revealing alterations in the way the molecules interact and the reaction products are formed.
Natural mutations alter the function of HSPA1A.
The goal of this project was to determine the functional output of natural occurring mutations (SNPs) on the HSPA1A gene in different human populations .
 Methods
I480N
 Future Work
Clone wild-type (WT) and mutant HSPA1A
Purify recombinant proteins from bacteria
Determine functional changes
by Isothermal Titration Calorimetry (ITC)
- Test in vivo whether the observed functional changes are biologically important.
F592S
 Acknowledgements
Figure 5: ITC analysis of WT and mutated HSPA1A variants with ADP(A), ATP (B), and protein substrate (C).
This work was supported by a State Mini-Grant to NN. We would also like to thank our lab mates in Nikolaidis lab as well as Dr. Rasche for allowing us to use the ITC machine.
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