Structure, function and dynamics of proteins by NMR spectroscopy : Application to human J-Protein co-chaperone Dph4 and yeast Tim23-IMS

Abstract

Research Focus This research explores the application of NMR spectroscopy in protein biochemistry, specifically in:

Structure determination of human J-protein co-chaperone Dph4 and yeast Tim23.

Functional analysis of human Dph4 using biophysical and biochemical techniques.

Characterization of hydrogen exchange in proteins using ^13C-detected 2D CON experiments.

Chapter 1 - Introduction Scope of NMR spectroscopy in protein structure determination.

Advantages of NMR over X-ray crystallography for natively unfolded and multi-domain proteins.

Residue-specific insights into dynamics, conformational exchange, and allostery.

Overview of human Dph4 (a type III J-protein) and yeast Tim23 (a mitochondrial translocon component).

Development of new NMR methods for solvent-exchangeable proton analysis.

Chapter 2 - Functional Analysis of Human Dph4 J-proteins are obligate partners of Hsp70s, stimulating ATPase activity.

Dph4, a type III J-protein, shows unique iron-binding ability with 16-fold higher affinity than zinc.

Fe-Dph4 undergoes oligomerization, potentially functioning as a transient iron-storage protein.

Exhibits redox and electron carrier activity critical for diphthamide biosynthesis.

Fe-binding stabilizes Dph4 and enhances its Hsp70-dependent functions.

Conserved Fe-binding property observed in yeast ortholog Jjj3.

Chapter 3 - Structural Analysis of Human Dph4 NMR structure of Zn-Dph4 solved.

Protein consists of an N-terminal helical domain and a C-terminal -sheet domain connected by a helical linker.

Domains fold independently but show inter-domain mobility.

Linker undergoes conformational changes leading to open/closed states; closed conformation stabilizes Hsp70:Dph4 interaction.

Chemical shift differences highlight the importance of Fe-binding in functional regulation.

Chapter 4 - Structural Analysis of Yeast Tim23 (IMS Domain) Tim23 is essential for mitochondrial protein import.

NMR analysis of IMS domain revealed large intrinsically disordered regions with a single helical region.

Provides first structural insights into the molecular details of mitochondrial translocation machinery.

Chapter 5 - NMR-Based Hydrogen Exchange Studies Developed a rapid method using modified ^13C-detected 2D CON experiments.

Enables simultaneous measurement of exchange rates and fractionation factors.

Illustrated on three proteins: Tim23 IMS-domain, ubiquitin, and a Dph4 truncation mutant.

Method provides efficient characterization of hydrogen exchange kinetics and thermodynamics.