Biochemistry Online: An Approach Based on Chemical Logic

CHAPTER 2 - PROTEIN STRUCTURE

D: PROTEIN FOLDING AND STABILITY

BIOCHEMISTRY - DR. JAKUBOWSKI

Last Update: 3/1/16

Learning Goals/Objectives for Chapter 2D: After class and this reading, students will be able to

differentiate between thermodynamic (equilibrium) and kinetic (timed) approaches to the study of protein folding reactions
describe techniques to study transient (kinetic) and long-lived (thermodynamic) intermediates in protein folding
describe the following intermediates in protein folding: molten globule, X-Pro isomers; Disulfide bond intermediates
interpret spectral and chromatographic data from protein folding studies and use this to determine or explain a mechanism for folding
describe properties of folded, unfolded, molten globule, and intrinsically disordered proteins
explain the difference between the environments for protein folding when performed in vitro and in vivo
state the role of molecular chaperones in in vivo protein folding
describe differences in disulfide bond occurrence in cytoplasmic and extracellular proteins

D4. The Denatured State

Although the structure of native and native-like states can be determined using x-ray crystallography and in solution using NMR, little detailed information exists on the actual structure of denatured and intermediate states. Intermediate states are difficult to trap in a way that allow details structural analysis. In contrast to the "native" state which consists of an ensemble of closely related states, intermediates and denatured states would consist of an ensemble of many different states, making structural analysis more difficult. Religa and others from Fersht's lab have engineered a mutant of the engrailed homeodomain (En-HD) from Drosophila melanogaster that allows such structural analyses to be performed. The mutation, Leu16Ala (L16A), destabilizes the protein such that it can be denatured simply by changing ionic strength. It is stable at high ionic strength and folds quickly under those condition. However at physiological ionic strength it is "denatured" but contains significant alpha-helical structure but has nonnative contacts. It behaves like an early folding intermediate in that if placed in solutions of higher ionic strength it rearranges to form the native state. If placed in lower ionic strength, it progressively "unfolds" to yet other states. Given the ambiguities in how to define denatured and early folding intermediates states, Ferscht's group suggest an "explicit" definition of the denatured state. They define the unfolded state (U) as the "maximally unfolded state of a protein, in which the backbone NH groups have little protection against 1H/2H exchange". They define the denatured state, D, as the "lowest energy non-native state under a defined set of conditions". In this scenario, the denatured state could also be a folding intermediate if placed in conditions that promote folding. Previous work from the group showed that the denatured state of En-HD has three helices protected from 2H exchange and was one kcal/mol lower in energy than the unfolded state.

Jmol: Updated Engrailed homeodomain - denatured and native state Jmol14 (Java) | JSMol (HTML5)

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