The mechanism of neurotransmitter release has been extensively characterized, showing that vesicle fusion is mediated by the SNARE complex formed by syntaxin‐1, SNAP‐25 and synaptobrevin. This complex is disassembled by N‐ethylmaleimide sensitive factor (NSF) and SNAPs to recycle the SNAREs, whereas Munc18‐1 and Munc13s organize SNARE complex assembly in an NSF‐SNAP‐resistant manner. Synaptotagmin‐1 acts as the Ca2+ sensor that triggers exocytosis in a tight interplay with the SNAREs and complexins. Here, we review technical aspects associated with investigation of protein interactions underlying these steps, which is hindered because the release machinery is assembled between two membranes and is highly dynamic. Moreover, weak interactions, which are difficult to characterize, play key roles in neurotransmitter release, for instance by lowering energy barriers that need to be overcome in this highly regulated process. We illustrate the crucial role that structural biology has played in uncovering mechanisms underlying neurotransmitter release, but also discuss the importance of considering the limitations of the techniques used, including lessons learned from research in our lab and others. In particular, we emphasize: (a) the promiscuity of some protein sequences, including membrane‐binding regions that can mediate irrelevant interactions with proteins in the absence of their native targets; (b) the need to ensure that weak interactions observed in crystal structures are biologically relevant; and (c) the limitations of isothermal titration calorimetry to analyze weak interactions. Finally, we stress that even studies that required re‐interpretation often helped to move the field forward by improving our understanding of the system and providing testable hypotheses.

Human dystrophin is a cytoplasmic protein that consists mainly of 24 spectrin domains positioned in tandem. Mutations in these highly conserved spectrin motifs are known to cause muscle degeneration, highlighting their role in maintaining the integrity of muscle fibers during movement. Through circular dichroism (CD), fluorescence spectroscopy (FS), and differential scanning calorimetry (DSC), the spectrins in their monomeric (S17), dimeric (S17-18), and trimeric (S17-19) forms were thermally denatured. Global fitting of CD and FS data, constrained with DSC, revealed a non-additive change in ΔGunfolding consistent with a negative coupling mechanism where the spectrins undergo mutual destabilization. This mechanism was further explored by means of Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR) studies with the hypothesis that the addition of spectrin repeats confers increased structural disorder. EPR scans of S17 and S17-19 show that the trimer experiences a higher degree of mobility as compared to the monomer, supporting our hypothesis. Additionally, we will continue to investigate the conformational disorder and overall stability of hydrated spectrin constructs through Overhauser Dynamic Nuclear Polarization (ODNP), which will provide a better understanding of the local water matrix and spatial heterogeneity surrounding the hydration layer of the spectrins. From the cumulative spectroscopic data obtained through these integrated approaches, we aim to derive a more comprehensive understanding of dystrophin's elusive mechanism.

Spectrin repeat domains are a highly conserved biological motif found in many human structural proteins. Dystrophin contains 24 tandem spectrin repeats which provide structural support through mediating interactions between intracellular actin filaments and the extracellular matrix. However, the molecular mechanism by which dystrophin provides this support is unknown. Understanding this underlying structure/function relationship is important because mutations in dystrophin directly cause muscular dystrophy. Thus far, the following constructs have been expressed in E. coli, purified using chromatography, and thermodynamically characterized: S17, S17-18, S17-19. Parameters were determined through globally fitting thermal denaturation signals from Fluorescence Spectroscopy (FS), Circular Dichroism (CD), and Fluorescence Lifetime Spectroscopy (FLT) to a two-state model of unfolding. Fits were constrained using ΔCp values determined using Differential Scanning Calorimetry (DSC). This parameterization then allowed for determination of the free energy of stability (ΔGunfolding) of each construct. Results indicate that the ΔGunfolding of S17 is nearly double that of S17-19. This pronounced non-additivity indicates that tandem spectrin repeats mutually destabilize each other, termed negative coupling. Additionally, the comparison of Electron Paramagnetic Resonance (EPR) spectra of S17 and S17-19 indicate that the trimer exhibits greater local confirmational flexibility, consistent with decreased stability. To further test this negative coupling hypothesis, we are purifying S19 for thermodynamic characterization. This will allow for the comparison of the sum of S19 and S17-18's ΔGunfolding values with that of the trimer, helping further reveal the energetic and structural basis of dystrophin's mechanism.

While the thermodynamic signature of protein domain coupling from a computational as well as experimental standpoint exists, the underlying mechanistic details as to the underlying cause and functional consequences do not. Protein domain coupling is where one protein domain impacts the structural and functional properties of another domain of the protein. Human dystrophin protein is a means to elucidate the basis of protein domain coupling as the proteins involved in force transduction often have the common theme of being comprised of a series of tandem repeat protein domains. Furthermore, 24 of the 27 domains of dystrophin are spectrin repeats where spectrins are a protein domain occurring in myriad organisms and associated with a diversity of functions, raising additional questions as to whether coupling is a conserved feature of spectrins or unique to dystrophin in force transducing proteins. Using a combination of thermodynamic and structural dynamic methodologies, we seek to determine how observed coupling via thermodynamic analysis is manifested structurally in spectrin domains 17, 18 and 19 from Human dystrophin.

We have used circular dichroism, co-sedimentation and pulsed EPR to assess the structural and functional impact of a disease-causing mutation belonging to beta-III-spectrin within dystrophin's first actin-binding domain. Beta-III-spectrin and dystrophin have several commonalities including a structural preference for "closed" conformations of their calponin-homology domains as well as an apparent hydrophobic driving force for occupation of such closed conformations. Most of the known disease-causing mutations in dystrophin ABD1, however, cause misfolding with subsequent targeting of the protein to the proteasome, so it is difficult to use mutations to gain further insights into its structural mechanism for regulating actin binding. Here, we explore the possibility of a beta-III-spectrin missense mutation site that is strongly conserved in dystrophin's sequence as a potential tool to probe structure-function relationships of the dystrophin ABD1 that may parallel its close evolutionary relative.

We used time-resolved FRET, circular dichroism, and all-atom simulation to investigate the structural impact of phosphorylation and dielectric constant on synaptotagmin 1's intrinsically disordered region (IDR). We found that the full-length IDR sequence, a ∼60 residue strong polyampholyte that when studied in the form of a peptide, undergoes structural collapse consistent with its κ-predicted behavior. Furthermore, we found that the exocytosis-modulating phosphorylation of Thr112, a residue located in the IDR's more sequence diverse central core region, disrupts a local disorder-to-order transition that occurs when solution dielectric constant is lowered and helical structure is stabilized by addition of trifluoroethanol. Implicit solvent simulations testing the impact of dielectric constant alone converge on a similar result, showing that helical structure is formed with higher probability at a reduced dielectric, where several lysine-aspartic acid salt bridges stabilize transient secondary structure. Phosphorylation, however, results in formation of salt bridges unsuitable for helix formation. These results suggest a model where compaction of the IDR sequence and phosphorylation may regulate structural transitions that in turn modulate neuronal exocytosis.

We used time-resolved Förster resonance energy transfer, circular dichroism, and molecular dynamics simulation to investigate the structural dependence of synaptotagmin 1's intrinsically disordered region (IDR) on phosphorylation and dielectric constant. We found that a peptide corresponding to the full-length IDR sequence, a ∼60-residue strong polyampholyte, can sample structurally collapsed states in aqueous solution, consistent with its κ-predicted behavior, where κ is a sequence-dependent parameter that is used to predict IDR compaction. In implicit solvent simulations of this same sequence, lowering the dielectric constant to more closely mimic the environment near a lipid bilayer surface promoted further sampling of collapsed structures. We then examined the structural tendencies of central region residues of the IDR in isolation. We found that the exocytosis-modulating phosphorylation of Thr112 disrupts a local disorder-to-order transition induced by trifluoroethanol/water mixtures that decrease the solution dielectric constant and stabilize helical structure. Implicit solvent simulations on these same central region residues testing the impact of dielectric constant alone converge on a similar result, showing that helical structure is formed with higher probability at a reduced dielectric. In these helical conformers, lysine-aspartic acid salt bridges contribute to stabilization of transient secondary structure. In contrast, phosphorylation results in formation of salt bridges unsuitable for helix formation. Collectively, these results suggest a model in which phosphorylation and compaction of the IDR sequence regulate structural transitions that in turn modulate neuronal exocytosis.

Dystrophin, a 427 kDa protein located on one of the largest genes in the human genome, has been implicated in myocytes' ability to dissipate mechanical forces transduced between cytoskeletal and membrane features. Single point mutations in Dystrophin can result in myocyte membrane shearing under normal muscular flexion and is diagnosed as Becker's Muscular Dystrophy. Given point mutations outside of direct binding sites can render Dystrophin at a loss of function, the amino acids within each domain are likely stabilized or destabilized allosterically. Our research aims to determine how secondary and tertiary structures of Dystrophin can effectively couple during unfolding transitions within and between protein domains. Here, we study the actin binding domain (ABD1) of Dystrophin which contains the highest frequency of point mutations resulting in Becker's muscular dystrophy. In order to gain insight as to how ABD1 may couple within itself, we designed a series of thermodynamic analyses to describe ABD1 unfolding semi-mechanistically. We use differential scanning calorimetry as well as additional spectroscopic techniques (CD and FLT) to monitor ABD1 unfolding energetics and determine how energy may be stored in ABD1's secondary structure or in its buried hydrophobic residues. Relating these approaches provides a comparative approach as to how different structural features may affect ABD1's ability to dissipate mechanical stress through folding and unfolding thermodynamically favorable folding features.

We have used pulsed electron paramagnetic resonance, calorimetry, and molecular dynamics simulations to examine the structural mechanism of binding for dystrophin's N-terminal actin-binding domain (ABD1) and compare it to utrophin's ABD1. Like other members of the spectrin superfamily, dystrophin's ABD1 consists of two calponin-homology (CH) domains, CH1 and CH2. Several mutations within dystrophin's ABD1 are associated with the development of severe degenerative muscle disorders Duchenne and Becker muscular dystrophies, highlighting the importance of understanding its structural biology. To investigate structural changes within dystrophin ABD1 upon binding to actin, we labeled the protein with spin probes and measured changes in inter-CH domain distance using double-electron electron resonance. Previous studies on the homologous protein utrophin showed that actin binding induces a complete structural opening of the CH domains, resulting in a highly ordered ABD1-actin complex. In this study, double-electron electron resonance shows that dystrophin ABD1 also undergoes a conformational opening upon binding F-actin, but this change is less complete and significantly more structurally disordered than observed for utrophin. Using molecular dynamics simulations, we identified a hinge in the linker region between the two CH domains that grants conformational flexibility to ABD1. The conformational dynamics of both dystrophin's and utrophin's ABD1 showed that compact conformations driven by hydrophobic interactions are preferred and that extended conformations are energetically accessible through a flat free-energy surface. Considering that the binding free energy of ABD1 to actin is on the order of 6-7 kcal/mole, our data are compatible with a mechanism in which binding to actin is largely dictated by specific interactions with CH1, but fine tuning of the binding affinity is achieved by the overlap between conformational ensembles of ABD1 free and bound to actin.

Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the cytoskeletal protein β-III-spectrin. Previously, a SCA5 mutation resulting in a leucine-to-proline substitution (L253P) in the actin-binding domain (ABD) was shown to cause a 1000-fold increase in actin-binding affinity. However, the structural basis for this increase is unknown. Here, we report a 6.9 Å cryo-EM structure of F-actin complexed with the L253P ABD. This structure, along with co-sedimentation and pulsed-EPR measurements, demonstrates that high-affinity binding caused by the CH2-localized mutation is due to opening of the two CH domains. This enables CH1 to bind actin aided by an unstructured N-terminal region that becomes α-helical upon binding. This helix is required for association with actin as truncation eliminates binding. Collectively, these results shed light on the mechanism by which β-III-spectrin, and likely similar actin-binding proteins, interact with actin, and how this mechanism can be perturbed to cause disease.

We have used site-directed spectroscopy to examine disease-related structural transitions in the N-terminal actin binding domains (ABDs) of the proteins dystrophin and β-III-spectrin. Both ABDs contain tandem calponin homology (CH) domains that are implicated in disease, dystrophin in muscular dystrophy and spectrin in spinocerebellar ataxia type 5. By placing spin and fluorescent labels within each CH domain and measuring the microsecond- and nanosecond-resolved dipolar electron-electron resonance (DEER) and fluorescence resonance energy transfer (FRET), we found that dystrophin's CH domains transition from a compact closed conformation to a more open extended conformation upon complex formation with actin. In complementary studies, time-resolved phosphorescence anisotropy of actin reveals cooperative restriction of actin dynamics upon complex formation. Mutations in dystrophin that cause muscular dystrophy affect both measurements, suggesting that cooperative structural dynamics is intimately involved in disease progression. Molecular dynamics simulations indicate that structural transitions in dystrophin are regulated in part by hydrophobic interactions between CH domains. A disease-causing mutation within β-III-spectrin's ABD, which results in a 1000-fold increase in actin binding affinity, perturbs the natural hydrophobic contacts between its CH domains, and spectroscopic probes provide crucial insight into the molecular mechanism. We propose that dystrophin and β-spectrin proteins share a common mechanism of actin regulation, based on coupling between structural dynamics of actin and ABD. Funding to DDT (NIH grant AR63007). Funding to TSH (NIH grant RO1GM44757).

Synaptotagmin 1 (Syt 1) is an integral membrane protein responsible for sensing the calcium ion (Ca2+) influx in neurons that triggers synaptic vesicle exocytosis. How Syt 1's intrinsically disordered region (IDR), a ∼60 residue sequence located between the protein's transmembrane helix and two Ca2+-sensing C2 domains, contributes to protein function is not well understood. The same is true of analogous IDRs located in the other synaptotagmin isoforms. Recently, we found that the Syt 1 IDR is structurally responsive to vesicles whose lipid composition mimics that of a synaptic vesicle organelle and that this sensitivity allosterically influences binding and folding behavior of the adjacent C2 domain. We believe these observations may be applicable to the study of other synaptotagmin isoforms and discuss generally how an IDR-membrane interaction could contribute to modulation of C2 domain function.

Dystrophin is a filamentous muscle protein that links the cytoskeleton to the extracellular matrix, dampening the laterally transduced force of sarcomere shortening and lengthening and thus limiting the mechanical stress reaching the sarcolemma membrane. Dystrophin's homologue, utrophin, fulfills a similar role in developing muscle but is ultimately replaced by dystrophin. Previous spectroscopic measurements on full-length dystrophin and utrophin showed that each significantly restrict the amplitude of actin filament rotational dynamics upon binding, but utrophin restricts this motion to a much greater extent. We hypothesize that part of this functional difference between homologous proteins stems from distinct structural binding modes and dynamics in the first actin-binding domain (ABD1). Using double electron-electron resonance (DEER) and time-resolved FRET, we characterized the closed-to-open structural transition of dystrophin's ABD1 and compared it to the previously studied utrophin. Dystrophin exhibits more disorder and a greater propensity to be in a closed (compact) state. To understand and complement these spectroscopic findings, we tested the thermal stability of dystrophin's ABD1 and examined possible driving forces for ABD1 closure with molecular dynamics (MD) simulations. We found that dystrophin is only marginally stable, consistent with the structural disorder measured spectroscopically. In MD simulations, the ABD1 crystal structure relaxed to a closed compact structural state and thus protected several hydrophobic residues. This suggests that location of hydrophobic residues is an important dictator of dystrophin's open-closed structural equilibrium. Together these results indicate that dystrophin's ABD1 forms an unstable complex upon binding to F-actin, due to energetically unfavorable exposure of hydrophobic surfaces within ABD1. These features may contribute to the reduced overall restriction of actin dynamics in comparison to utrophin. Funding to DDT (NIH grant AR63007).

Synaptotagmin I (Syt I) is a vesicle-localized integral membrane protein that senses the calcium ion (Ca(2+)) influx to trigger fast synchronous release of neurotransmitter. How the cytosolic domains of Syt I allosterically communicate to propagate the Ca(2+) binding signal throughout the protein is not well understood. In particular, it is unclear whether the intrinsically disordered region (IDR) between Syt I's transmembrane helix and first C2 domain (C2A) plays an important role in allosteric modulation of Ca(2+) binding. Moreover, the structural propensity of this IDR with respect to membrane lipid composition is unknown. Using differential scanning and isothermal titration calorimetry, we found that inclusion of the IDR does indeed allosterically modulate Ca(2+) binding within the first C2 domain. Additionally through application of nuclear magnetic resonance, we found that Syt I's IDR interacts with membranes whose lipid composition mimics that of a synaptic vesicle. These findings not only indicate that Syt I's IDR plays a role in regulating Syt I's Ca(2+) sensing but also indicate the IDR is exquisitely sensitive to the underlying membrane lipids. The latter observation suggests the IDR is a key route for communication of lipid organization to the adjacent C2 domains.

We have used time-resolved EPR and fluorescence to resolve structural transitions of dystrophin upon actin binding. Dystrophin (Dys) is a muscle cytoskeletal protein that binds to filamentous actin (F-actin) and the dystroglycan complex in the sarcolemmal membrane. Dys acts to dissipate mechanical forces generated during the contraction and relaxation of muscle thereby maintaining sarcolemmal membrane integrity and protecting from tears. The protein-protein interactions and allostery underlying this function of Dys have not been well studied in the context of conformational change and thermodynamics, partly because acquisition of structural and thermodynamic detail on large and flexible proteins is difficult. Two techniques capable of measuring large-scale conformational changes are dipolar electron-electron resonance (DEER) and time-resolved fluorescence resonance energy transfer (TR-FRET). Using a combination of DEER and TR-FRET, we placed a single label (nitroxide or fluorescent) in each CH domain Dys ABD1 and subsequently measured the interprobe distance to assess conformational change upon association with F-actin. To probe the allosteric network of Dys ABD1, we also subjected the protein to differential scanning calorimetry.

Dystrophin and utrophin are homologous actin-binding proteins that link actin filaments to the sarcolemmal membrane, forming crucial contacts between the two cellular structures that maintain membrane integrity in muscle filaments. In several types of muscular dystrophy, dystrophin is mutated or truncated, disrupting its structural support role leading to membrane damage and ultimately progressive muscle weakening in afflicted individuals. The binding of dystrophin and utrophin proteins is mediated by actin binding domains (ABD) and in certain subtypes of muscular dystrophy, single point mutations in the first ABD are sufficient to disrupt the entire protein's function. While it is not clear how a single point mutation disrupts function so severely, more recent binding studies point to the importance of allosteric mechanisms in the protein's interaction with actin. To gain further insights into these mechanisms, we combined differential scanning calorimetry and time-resolved fluorescence spectroscopy in a thermal denaturation approach to thermodynamically dissect allostery within the ABD1 region of utrophin and dystrophin. Acknowledgements This work was supported by grant to DDT from the Muscular Dystrophy Association (MDA4322).

Synaptotagmin I (Syt I) contains two Ca2+ and phospholipid binding domains (C2A and C2B) that function in the regulated exocytosis of neurotranmsitters. Syt I participates in the sensing of the Ca2+ influx in nerve cells during signal propagation. C2A and C2B are linked to each other with C2A, the domain closest to the vesicle, having a linker region connecting the protein to a vesicle. A combination of differential scanning calorimetry and fluorescence lifetime spectroscopy were used to study free energy of stability of C2A constructs with varying length of the linker region. These studies showed a decrease in stability with the inclusion of the linker region. From these findings, we propose that the linker region has an impact on the binding affinity of endogenous ligands (phospholipids and Ca2+) by the binding domain. We used isothermal titration calorimetry to investigate and compare the binding of endogenous ligand by the domain with and without the linker. The inclusion of the linker led to a decrease in binding affinity, but the linker promoted cooperativity between binding sites allowing for an increased responsiveness to local ligand concentrations. Although the linker is not in close proximity to the Ca2+ binding loops, there is still an effect on binding. This can be linked to the relatively low stability, which allows for small structural changes to make a difference in the ligand binding. The cooperativity between binding sites allows for the saturation of C2A to be more dramatic over a narrower range of ligand concentration. The wider range of responsiveness suggests that Syt I is able to respond better to the influx of Ca2+ affecting its ability to participate in exocytotic events.

Synaptotagmin I is a protein involved in the final steps of neurotransmitter release assisting in the fusion of vesicles with the plasma membrane of nerve cells. The synaptotagmin protein consists of a single transmembrane region connected to two C2 domains, C2A and C2B, through a long unstructured linker region. While much work has been done in order to understand how synaptotagmin functions in the fusion process, it has largely been focused solely on the C2 domains of the protein, with little work done examining the linker region. Recently it has been shown that these domains show remarkably low stabilities, and that the two C2 domains are energetically coupled. These results paint a picture of a protein that is exquisitely sensitive to its environment. As such these findings have led us to hypothesize that the unstructured linker region that tethers the C2 domains to the membrane may play an important role in the function of synaptotagmin. In order to gain a better understanding on how the presence of this unstructured region impacts synaptotagmin, we have investigated the stability of the C2A domain both with and without the linker region and have found dramatic differences between the two constructs. We have found that under all ligand conditions the stability of the domain decreases. For example, in the presence of Ca2+ the stability of the C2A domain with the linker region was found to be 3.61±0.03 (kcal/mol) compared to a stability of 4.32±0.05 (kcal/mol) for the domain without the linker. The decrease found in the stability of domain when connected with the linker region suggests that this region acts to increase the sensitivity of the C2A domain to its environment, and as such may tune the responsiveness of the domain.