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Differential scanning fluorimetry (DSF) using the inherent fluorescence of proteins (nDSF) is a popular technique to evaluate thermal protein stability in different conditions (e.g. buffer, pH). In many cases, ligand binding increases thermal stability of a protein and often this can be detected as a clear shift in nDSF experiments. Here, we evaluate binding affinity quantification based on thermal shifts. We present four protein systems with different binding affinity ligands, ranging from nM to high μM. Our study suggests that binding affinities determined by isothermal analysis are in better agreement with those from established biophysical techniques (ITC and MST) compared to apparent K d s obtained from melting temperatures. In addition, we describe a method to optionally fit the heat capacity change upon unfolding ( Δ C p ) during the isothermal analysis. This publication includes the release of a web server for easy and accessible application of isothermal analysis to nDSF data.

The solution and crystal structures of a bacterial phytochrome photosensory core in both its resting and activated states are determined; switching between closed (resting) and open (activated) forms is found to be mediated by a conserved ‘tongue’, and the structures indicate that smaller changes in the vicinity of the chromophore are amplified in scale as they are transmitted through the tongue and beyond. Signal amplification by light-receptor proteins The crystal structure of the photosensory core of a bacterial phytochrome in both the resting and the active (illuminated) state has now been solved. Working with the phytochrome from the extremophile Deinococcus radiodurans , Sebastian Westenhoff and colleagues demonstrate that toggling between resting and active forms is mediated by a conserved 'tongue' that contacts the chromophore. Atomic-scale structural changes in the vicinity of the chromophore are amplified as they are transmitted through the tongue and beyond, culminating in a nanometre-scale conformational signal that feeds into the rest of the cellular signalling network. Sensory proteins must relay structural signals from the sensory site over large distances to regulatory output domains. Phytochromes are a major family of red-light-sensing kinases that control diverse cellular functions in plants, bacteria and fungi 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 . Bacterial phytochromes consist of a photosensory core and a carboxy-terminal regulatory domain 10 , 11 . Structures of photosensory cores are reported in the resting state 12 , 13 , 14 , 15 , 16 , 17 , 18 and conformational responses to light activation have been proposed in the vicinity of the chromophore 19 , 20 , 21 , 22 , 23 . However, the structure of the signalling state and the mechanism of downstream signal relay through the photosensory core remain elusive. Here we report crystal and solution structures of the resting and activated states of the photosensory core of the bacteriophytochrome from Deinococcus radiodurans . The structures show an open and closed form of the dimeric protein for the activated and resting states, respectively. This nanometre-scale rearrangement is controlled by refolding of an evolutionarily conserved ‘tongue’, which is in contact with the chromophore. The findings reveal an unusual mechanism in which atomic-scale conformational changes around the chromophore are first amplified into an ångstrom-scale distance change in the tongue, and further grow into a nanometre-scale conformational signal. The structural mechanism is a blueprint for understanding how phytochromes connect to the cellular signalling network.

During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process in yeast, endocytic coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryo-EM structure of a 16-mer complex of the ANTH and ENTH membrane-binding domains from Sla2 and Ent1 bound to PIP 2 that constitutes the anchor to the plasma membrane. Detailed in vitro and in vivo mutagenesis of the complex interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate its biological relevance. A hetero-tetrameric unit binds PIP 2 molecules at the ANTH-ENTH interfaces and can form larger assemblies to contribute to membrane remodeling. Finally, a time-resolved small-angle X-ray scattering study of the interaction of these adaptor domains in vitro suggests that ANTH and ENTH domains have evolved to achieve a fast subsecond timescale assembly in the presence of PIP 2 and do not require further proteins to form a stable complex. Together, these findings provide a molecular understanding of an essential piece in the molecular puzzle of clathrin-coated endocytic sites. During clathrin-mediated endocytosis in yeast, endocytic coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for membrane invagination. Here authors present a cryo-EM structure of a 16-mer complex of the ANTH and ENTH membrane-binding domains from Sla2 and Ent1 bound to PIP 2 that constitutes the anchor to the plasma membrane.

Members of the SH3- and ankyrin repeat (SHANK) protein family are considered as master scaffolds of the postsynaptic density of glutamatergic synapses. Several missense mutations within the canonical SHANK3 isoform have been proposed as causative for the development of autism spectrum disorders (ASDs). However, there is a surprising paucity of data linking missense mutation-induced changes in protein structure and dynamics to the occurrence of ASD-related synaptic phenotypes. In this proof-of-principle study, we focus on two ASD-associated point mutations, both located within the same domain of SHANK3 and demonstrate that both mutant proteins indeed show distinct changes in secondary and tertiary structure as well as higher conformational fluctuations. Local and distal structural disturbances result in altered synaptic targeting and changes of protein turnover at synaptic sites in rat primary hippocampal neurons.

Sensor histidine kinases are central to sensing in bacteria and in plants. They usually contain sensor, linker, and kinase modules and the structure of many of these components is known. However, it is unclear how the kinase module is structurally regulated. Here, we use nano- to millisecond time-resolved X-ray scattering to visualize the solution structural changes that occur when the light-sensitive model histidine kinase YF1 is activated by blue light. We find that the coiled coil linker and the attached histidine kinase domains undergo a left handed rotation within microseconds. In a much slower second step, the kinase domains rearrange internally. This structural mechanism presents a template for signal transduction in sensor histidine kinases. Sensor histidine kinases (SHK) consist of sensor, linker and kinase modules and different models for SHK signal transduction have been proposed. Here the authors present nano- to millisecond time-resolved X-ray scattering measurements, which reveal a structural mechanism for kinase domain activation in SHK.

This work demonstrates a new method for investigating time-resolved structural changes in protein conformation and oligomerization via photocage-initiated time-resolved X-ray solution scattering by observing the ATP-driven dimerization of the MsbA nucleotide-binding domain. Photocaged small molecules allow the observation of single-turnover reactions of non-naturally photoactivatable proteins. The kinetics of the reaction can be derived from changes in X-ray scattering associated with ATP-binding and subsequent dimerization. This method can be expanded to any small-molecule-driven protein reaction with conformational changes traceable by X-ray scattering where the small molecule can be photocaged.

Calmodulin (CaM) is a very conserved, ubiquitous, eukaryotic protein that binds four Ca2+ ions with high affinity. It acts as a calcium sensor by translating Ca2+ signals into cellular processes such as metabolism, inflammation, immune response, memory, and muscle contraction. Calcium binding to CaM leads to conformational changes that enable Ca2+/CaM to recognize and bind various target proteins with high affinity. The binding mode and binding partners of CaM are very diverse, and a consensus binding sequence is lacking. Here, we describe an elegant system that allows conformation-specific detection of CaM-binding to its binding partners. We incorporate the unnatural amino acid p-azido-phenylalanine (AzF) in different positions of CaM and follow its unique spectral signature by infrared (IR)-spectroscopy of the azido stretching vibration. Our results suggest that the AzF vibrational probe is sensitive to the chemical environment in different CaM/CaM-binding domain (CaMBD) complexes, which allows differentiating between different binding motifs according to the spectral characteristics of the azido stretching mode. We corroborate our results with a crystal structure of AzF-labelled CaM (CaM108AzF) in complex with a binding peptide from calmodulin-dependent protein kinase IIα identifying the structural basis for the observed IR frequency shifts.

An essential element of adaptive immunity is selective binding of peptide antigens by major histocompatibility complex (MHC) class I proteins and their presentation to cytotoxic T lymphocytes. Using native mass spectrometry, we analyze the binding of peptides to an empty disulfide-stabilized HLA-A*02:01 molecule and, due to its unique stability, we determine binding affinities of complexes loaded with truncated or charge-reduced peptides. We find that the two anchor positions can be stabilized independently, and we further analyze the contribution of additional amino acid positions to the binding strength. As a complement to computational prediction tools, our method estimates binding strength of even low-affinity peptides to MHC class I complexes quickly and efficiently. It has huge potential to eliminate binding affinity biases and thus accelerate drug discovery in infectious diseases, autoimmunity, vaccine design, and cancer immunotherapy.

To interpret and transmit biological signals, proteins use correlated motions. Experimental determination of these dynamics and the structural distributions they generate remains a key challenge. Here, using 1146 crystal structures of the main protease (M pro ) from SARS-CoV-2, we were able to infer a model of the enzyme’s structural fluctuations. M pro is regulated by concentration, becoming enzymatically active after forming a homodimer. To understand the structural changes that enable dimerization to activate catalysis, we employed our model, predicting which regions of the dimerization domain are structurally correlated with the active site. Mutations at these positions, expected to disrupt catalysis, resulted in a dramatic reduction in activity in one case, a mild effect in the second, and none in the third. Additional crystallography and biophysical experiments provide a mechanistic explanation for these results. Our work suggests that a statistical crystallography, in which numerous crystallographic datasets are analyzed, can reveal the structural fluctuations of protein native states and help uncover their biological function. Using the small differences across 1146 crystal structures we collected of the SARS-CoV-2 main protease, we map correlated atomic fluctuations to predict and experimentally confirm a functional allosteric network.

An essential element of adaptive immunity is selective binding of peptide antigens by major histocompatibility complex (MHC) class I proteins and their presentation to cytotoxic T lymphocytes. Using native mass spectrometry, we analyze the binding of peptides to an empty disulfide-stabilized HLA-A*02:01 molecule and, due to its unique stability, we determine binding affinities of complexes loaded with truncated or charge-reduced peptides. We find that the two anchor positions can be stabilized independently, and we further analyze the contribution of additional amino acid positions to the binding strength. As a complement to computational prediction tools, our method estimates binding strength of even low-affinity peptides to MHC class I complexes quickly and efficiently. It has huge potential to eliminate binding affinity biases and thus accelerate drug discovery in infectious diseases, autoimmunity, vaccine design, and cancer immunotherapy. The authors present a sensitive and rapid method to determine the binding strength of MHC class 1 peptide complexes using native mass spectrometry.