Monitoring protein unfolding transitions by NMR-spectroscopy

NMR-spectroscopy has certain unique advantages for recording unfolding transitions of proteins compared e.g. to optical methods. It enables per-residue monitoring and separate detection of the folded and unfolded state as well as possible equilibrium intermediates. This allows a detailed view on the state and cooperativity of folding of the protein of interest and the correct interpretation of subsequent experiments. Here we summarize in detail practical and theoretical aspects of such experiments. Certain pitfalls can be avoided, and meaningful simplification can be made during the analysis. Especially a good understanding of the NMR exchange regime and relaxation properties of the system of interest is beneficial. We show by a global analysis of signals of the folded and unfolded state of GB1 how accurate values of unfolding can be extracted and what limits different NMR detection and unfolding methods. E.g. commonly used exchangeable amides can lead to a systematic under determination of the thermodynamic protein stability. We give several perspectives of how to deal with more complex proteins and how the knowledge about protein stability at residue resolution helps to understand protein properties under crowding conditions, during phase separation and under high pressure.

Claiming therapeutic protein stability in a clinical setting based on limited analytical data is misleading and dangerous

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Conformational Flexibility and Local Frustration in the Functional States of the SARS-CoV-2 Spike B.1.1.7 and B.1.351 Variants: Mutation-Induced Allosteric Modulation Mechanism of Functional Dynamics and Protein Stability

The experimental and computational studies of the SARS-CoV-2 spike protein variants revealed an important role of the D614G mutation that is shared across variants of concern(VOCs), linking the effect of this mutation with the enhanced virus infectivity and transmissibility. The recent structural and biophysical studies characterized the closed and open states of the B.1.1.7 (B.1.1.7) and B.1.351 (Beta) spike variants allowing for a more detailed atomistic characterization of the conformational landscapes and functional changes. In this study, we employed coarse-grained simulations of the SARS-CoV-2 spike variant trimers together with the ensemble-based mutational frustration analysis to characterize the dynamics signatures of the conformational landscapes. By combining the local frustration analysis of the conformational ensembles with collective dynamics and residue-based mutational scanning of protein stability, we determine protein stability hotspots and identify potential energetic drivers favoring the receptor-accessible open spike states for the B.1.1.7 and B.1.351 spike variants. Through mutational scanning of protein stability changes we quantify mutational adaptability of the S-G614, S-B.1.1.7 and S-B.1.351 variants in different functional forms. Using this analysis, we found a significant conformational and mutational plasticity of the open states for all studied variants. The results of this study suggest that modulation of the energetic frustration at the inter-protomer interfaces can serve as a mechanism for allosteric couplings between mutational sites, the inter-protomer hinges of functional motions and motions of the receptor-binding domain required for binding of the host cell receptor. The proposed mechanism of mutation-induced energetic frustration may result in the greater adaptability and the emergence of multiple conformational substates in the open form. This study also suggested functional relationships between mutation-induced modulation of protein dynamics, local frustration and allosteric regulation of the SARS-CoV-2 spike protein.

Cellular ATP Levels Determine the Stability of a Nucleotide Kinase

The energy currency of the cell ATP, is used by kinases to drive key cellular processes. However, the connection of cellular ATP abundance and protein stability is still under investigation. Using Fast Relaxation Imaging paired with alanine scanning and ATP depletion experiments, we study the nucleotide kinase (APSK) domain of 3′-phosphoadenosine-5′-phosphosulfate (PAPS) synthase, a marginally stable protein. Here, we show that the in-cell stability of the APSK is determined by ligand binding and directly connected to cellular ATP levels. The observed protein stability change for different ligand-bound states or under ATP-depleted conditions ranges from ΔGf0 = -10.7 to +13.8 kJ/mol, which is remarkable since it exceeds changes measured previously, for example upon osmotic pressure, cellular stress or differentiation. The results have implications for protein stability during the catalytic cycle of APS kinase and suggest that the cellular ATP level functions as a global regulator of kinase activity.

The Landscape-Based Protein Stability Analysis and Network Modeling of Multiple Conformational States of the SARS-CoV-2 Spike D614 Mutant: Conformational Plasticity and Frustration-Driven Allostery as Energetic Drivers of Highly Transmissible Spike Variant

The structural and functional studies of the SARS-CoV-2 spike protein variants revealed an important role of the D614G mutation that is shared across many variants of concern(VOCs), suggesting the effect of this mutation on the enhanced virus infectivity and transmissibility. The recent structural and biophysical studies provided important evidence about multiple conformational substates of the D614G spike protein. The development of a plausible mechanistic model which can explain the experimental observations from a more unified thermodynamic perspective is an important objective of the current work. In this study, we employed efficient and accurate coarse-grained simulations of multiple structural substates of the D614G spike trimers together with the ensemble-based mutational frustration analysis to characterize the dynamics signatures of the conformational landscapes. By combining the local frustration profiling of the conformational states with residue-based mutational scanning of protein stability and network analysis of allosteric interactions and communications, we determine the patterns of mutational sensitivity in the functional regions and sites of variants. We found that the D614G mutation may induce a considerable conformational adaptability of the open states in the SARS-CoV-2 spike protein without compromising folding stability and integrity of the spike protein. The results suggest that the D614G mutant may employ a hinge-shift mechanism in which the dynamic couplings between the site of mutation and the inter-protomer hinge modulate the inter-domain interactions, global mobility change and the increased stability of the open form. This study proposes that mutation-induced modulation of the conformational flexibility and energetic frustration at the inter-protomer interfaces may serve as an efficient mechanism for allosteric regulation of the SARS-CoV-2 spike proteins.