Fast-MAS Solid-State NMR as a Bypass to High-Molecular-Weight Proteins in Solution

Descripción del proyecto

Investigación de la dinámica de la columna vertebral de las proteínas de gran masa en solución

Las proteínas están formadas por aminoácidos unidos en una cadena. El movimiento de la «columna vertebral» de la proteína es fundamental para establecer su forma tridimensional y, por tanto, su actividad. Dado el papel decisivo de la dinámica de la columna vertebral de las proteínas en casi todas las funciones biológicas, estas han sido objeto de una intensa labor de investigación. Los métodos de resonancia magnética nuclear (RMN) más sensibles para abordar esta cuestión se ven actualmente limitados por restricciones de masa, lo que hace que las grandes proteínas más complejas y sus interacciones sean inaccesibles. En el proyecto bypassNMR, financiado por el Consejo Europeo de Investigación, se superará esta barrera con una combinación sin precedentes de RMN en estado sólido y RMN en disolución, lo que permitirá multiplicar por dos el rango de pesos moleculares accesible.

Objetivo

My objective is to establish methodology expanding the detailed characterization and exploitation of backbone dynamics in complex proteins up to 80-100 kDa monomer molecular weight. Experimental elucidation of protein motion is imperative for fundamental understanding of enzymatic and regulatory features. However, with a limit of regularly around 40-50 kDa maximum total mass, the more complex targets of current scientific interest usually evade solution NMR backbone resonance assignment and remain inaccessible for the majority of sophisticated methods for protein dynamics. This paradigmatic shortcoming has led to serious limitations in the understanding and exploitation of protein dynamics.
Here I aim to achieve a two-fold expansion of the accessible molecular-weight range by an unprecedented hybrid strategy. Based on the unmatched prospects of 4D and 5D solid-state NMR (ssNMR) assignment data for a 2x72 kDa protein, I will establish proton-detected, higher-dimensionality ssNMR methodology as a powerful framework for NMR assignment in an unprecedented size range. Subsequently, developing strategies utilizing ssNMR assignments as a springboard to solution NMR will enable detailed characterization of those targets under close-to-physiological conditions. This fundamentally new BYPASS strategy will allow understanding of intramolecular regulatory circuits and coupled motional networks in innumerable, previously inaccessible complex proteins, with a transformative impact for dynamics, in particular allosteric regulation, in structural biology.
Fueled by my role as a key player in revolutionizing solid-state NMR via proton-detected, fast magic-angle spinning NMR methodology, my achievements will be paradigmatic for the accessibility and utility of dynamics for the structure-dynamics-function relationship of proteins and will have widespread consequences for a wide range of structural biology and downstream applications such as pharmacology and biotechnology.