Energetic and kinetic folding mechanisms of the parallel β-helix protein pectate lyase C

Abstract

Pectate lyase C (peIC) was the first protein in which the parallel β-helix structure was recognized. The unique features of parallel β-helix-containing proteins - a simple topology and unusual interactions among side-chains - make peIC an interesting protein to study with respect to protein folding. In chapter 2 I discuss studies on the unfolding equilibrium of peIC. Gdn-HCI titrations monitored by far- and near-UV CD, and fluorescence spectroscopies were used to monitor the isothermal denaturation of peIC. Differential scanning calorimetry and temperature dependent CD were used to observe unfolding as a function of temperature. A detailed thermodynamic analysis of the denaturation was completed. In Chapter 3 the effects of the binding of anions to acid-unfolded peIC is presented. Addition of ANS to acid-denatured peIC leads to a large increase in the fluorescence quantum yield. This results from the induction of a partially folded protein species upon binding ANS. Thus, ANS does not act as a probe to detect partially folded species, but induces them. The mechanism of ANS binding and structure induction was studied. A study of the folding and unfolding kinetics is presented in Chapters 4 and 5. Three kinetic folding phases are observed with far-UV CD and four are observed with near-UV CD. The two slowest phases are the result of cis-trans isomerization of prolyl-peptide bonds. The fastest folding phase involves the formation of most, if not all, of the secondary structure and some tertiary structure. There is one kinetic phase observed with near-UV CD that has no corresponding far-UV CD phase. Mutagenesis results indicate that the slowest phase results from isomerization of the Leu219-Pro220 peptide bond and the intermediate slow phase is a result of the simultaneous isomerizations of multiple prolyl-peptide bonds. Quantum-chemical calculations on the CD spectrum of peIC are described in Chapter 6. Calculations on peIC with each individual tyrosine or tryptophan mutated to alanine were carried out and the difference spectra between the wild type and mutant proteins were obtained. A more complete structural model of the folding of peIC was deduced. It is proposed that the β-helix domain is formed in the fast kinetic phase.