Stabilization of a pH-sensitive apoptosis-linked coiled coil through single point mutations.
The apoptosis-associated Par-4 protein has been implicated in cancers of the prostate, colon, and kidney, and in Alzheimer's and Huntington's diseases, among other neurodegenerative disorders. Previously, we have shown that a peptide from the Par-4 C-terminus, which is responsible for Par-4 self-association as well as interaction with all currently identified effector molecules, is natively unfolded at neutral pH, but forms a tightly associated coiled coil at acidic pH and low temperature. Here, we have alternately mutated the two acidic residues predicted to participate in repulsive electrostatic interactions at the coiled coil interhelical interface. Analysis of circular dichroism spectra reveals that a dramatic alteration of the folding/unfolding equilibrium of this peptide can be effected through directed-point mutagenesis, confirming that the two acidic residues are indeed key to the pH-dependent folding behavior of the Par-4 coiled coil, and further suggesting that alleviation of charge repulsion through exposure to either a low pH microenvironment or an electrostatically complementary environment may be necessary for efficient folding of the Par-4 C-terminus.
Dutta
Kaushik
K
Department of Biochemistry and Biophysics, University of Rochester Medical Center, New York 14642, USA. dutta@nysbc.org
Engler
Frank A
FA
Cotton
Levaughn
L
Alexandrov
Andrei
A
Bedi
Gurrinder S
GS
Colquhoun
Jennifer
J
Pascal
Steven M
SM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D055672:Static Electricity; D013696:Temperature; D013816:Thermodynamics
D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C101296:prostate apoptosis response-4 protein