Emerging Approaches to Investigating Functional Protein Dynamics in Modular Redox Enzymes: Nitric Oxide Synthase as a Model System.

Abstract

Approximately 80% of eukaryotic and 65% of prokaryotic proteins are composed of multiple folding units (i.e., domains) connected by flexible linkers. These dynamic protein architectures, facilitated by linker regions, support essential functions such as electron transfer, respiration, and biosynthesis. This review critically assesses recent advancements in methods for studying protein dynamics, with a particular focus on modular, multidomain nitric oxide synthase (NOS) enzymes. Moving beyond traditional static "snapshots" of protein structures, current research emphasizes the dynamic nature of proteins, viewing them as flexible architectures modulated by conformational changes and interactions. In this context, the review discusses key developments in the integration of quantitative crosslinking mass spectrometry (qXL MS) with AlphaFold 2 predictions, which provides a powerful approach to disentangling NOS structural dynamics and understanding their modulation by external regulatory cues. Additionally, advances in site-specific infrared (IR) spectroscopy offer exciting potential in providing rich details about the conformational dynamics of NOSs in docked states. Moreover, optimization of genetic code expansion machinery enables the generation of genuine phosphorylated NOS enzymes, allowing detailed biophysical and functional analysis of phosphorylation's role in shaping NOS activity and structural flexibility; notably, this approach also empowers site-specific IR probe labeling with cyano groups. By embracing and leveraging artificial intelligence-driven tools like AlphaFold 2 for structural and conformational modeling, alongside solution-based biophysical methods such as site-specific IR spectroscopy and qXL MS, researchers will gain integrative insights into functional protein dynamics. Collectively, these breakthroughs highlight the transformative potential of modern approaches in driving fundamental biological chemistry research.