University of Cambridge
Stability, folding and evolution of the tumour suppressor protein p16.
Abstract
dc:description.abstractThe tumour suppressor protein, p16, is a member of the INK4 family of inhibitors of the cyclin D-dependent kinases 4 and 6 in the pRb pathway of eukaryotic cell cycle. The 156-amino acid protein consists of four ankyrin (ANK) repeats. The ANK repeat is a ubiquitous 33-residue sequence motif that adopts a β-hairpin-helix-turn-helix fold. Multiple repeats stack in a linear manner to produce an elongated structure that is stabilized predominantly by short-range interactions between residues. Purely composed of four ANK repeats, p16 is the minimal ANK-fold molecule. The thermodynamic and kinetic properties of p16 are unusual. The protein has a very low free energy of unfolding of 3.1 kcal mol-1 and a very rapid rate of unfolding of 0.8 s'1 at 25°C in the absence of denaturant. Thus, p16 is thermodynamically and kinetically unstable and is highly vulnerable to single point mutations. The transition-state structure of folding/unfolding of p16 is elucidated in this thesis. It is the first ANK-repeat protein to be studied in this way. The protein folds in two distinct parts. The two N-terminal ANK repeats that are distorted from the canonical structure in all of the INK4 proteins are unstructured, whilst the two C-terminal repeats are fully formed. The protein is commonly mutated in human tumours, frequently with missense substitutions. The mechanisms of inactivation of p16 by tumour-associated missense mutations were dissected using a systematic method designed to probe the integrity of the secondary structure and the global fold. They are classified into three categories: disruption of structural scaffold, distortion of CDK4/6-binding sites and reduction in thermodynamic stability. The coding sequence of p16 is exceptionally rich in CpG dinucleotides (almost eight times the average mammalian frequency), which may be one of the causes of the genetic instability of p16. The roles of the protein instability and genetic instability in the process of tumourigenesis are discussed. The relationship between the stability, folding and evolution of p16 substantiated by the findings of this study could provide a possible explanation for the existence of the plethora of tumour-associated mutations throughout this protein sequence.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2001
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Tang, Kit Shing.