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Single-molecule proteomic studies are critically important for understanding the molecular origins of cellular phenotypes. However, no currently available technology can achieve both the single-molecule sensitivity and high dynamic range required to comprehensively analyze the complex mixtures of proteins in biological samples. One approach to achieve high sensitivity across a wide dynamic range would be to create a protein array that arranges billions of single molecules with regular spacing on a patterned surface. However, creating such a protein array has remained an unsolved challenge for the field. Here, we present a highly scalable method for fabricating dense single-molecule protein arrays using a specially designed DNA origami structure, protein click-conjugation, photolithography and surface functionalization. The origami-structure is enhanced via terminal deoxynucleotidyl transferase-extension, which generates brush-like projections, increasing the effective size of the origami from 88 nm to greater than 200 nm. These particles are large enough to enable super-Poisson deposition of individual protein molecules on a nano-patterned chip (>98% occupancy with only 1% of sites occupied with multiple protein molecules). This approach allowed for single-molecule protein display of 600 million protein molecules per microscope slide-sized chip with the potential to scale further with denser feature spacing. We hypothesize that this technology will ultimately enable the development of highly scalable proteomic analysis platforms that address the currently unmet need for protein measurements at single-molecule sensitivity across an exceptionally wide dynamic range of protein concentrations.

Receptor clustering plays a key role in triggering cellular activation, but the relationship between the spatial configuration of clusters and the elicitation of downstream intracellular signals remains poorly understood. We developed a DNA-origami-based system that is easily adaptable to other cellular systems and enables rich interrogation of responses to a variety of spatially defined inputs. Using a chimeric antigen receptor (CAR) T cell model system with relevance to cancer therapy, we studied signaling dynamics at single-cell resolution. We found that the spatial arrangement of receptors determines the ligand density threshold for triggering and encodes the temporal kinetics of signaling activities. We also showed that signaling sensitivity of a small cluster of high-affinity ligands is enhanced when surrounded by nonstimulating low-affinity ligands. Our results suggest that cells measure spatial arrangements of ligands, translate that information into distinct signaling dynamics, and provide insights into engineering immunotherapies.

SUMMARYT cell receptor clustering plays a key role in triggering cell activation, but the relationship between the spatial configuration of clusters and elicitation of downstream intracellular signals remains poorly understood. We developed a DNA-origami-based system that is easily adaptable to other cellular systems and enables rich interrogation of responses to a variety of spatially defined inputs. Using a chimeric antigen receptor (CAR) T cell model system with relevance to cancer therapy, we studied signaling dynamics at single cell resolution. We found that the spatial arrangement of receptors determines the ligand density threshold for triggering and encodes the temporal kinetics of signaling activities. We also showed that signaling sensitivity of a small cluster of high-affinity ligands is enhanced when surrounded by non-stimulating low-affinity ligands. Our results suggest that cells measure spatial arrangements of ligands and translates that information into distinct signaling dynamics, and provide insights into engineering new immunotherapies.

Correct reconstruction of macromolecular structure by cryo-electron microscopy (cryo-EM) relies on accurate determination of the orientation of single-particle images. For small (, Editor’s summary: DNA origami orients single proteins on a surface to aid structure determination by cryo-electron microscopy.

A collection of programs is presented to analyze the thermodynamics of folding of linear repeat proteins using a 1D Ising model to determine intrinsic folding and interfacial coupling free energies. Expressions for folding transitions are generated for a series of constructs with different repeat numbers and are globally fitted to transitions for these constructs. These programs are designed to analyze Ising parameters for capped homopolymeric consensus repeat constructs as well as heteropolymeric constructs that contain point substitutions, providing a rigorous framework for analysis of the effects of mutation on intrinsic and directional (i.e., N- versus C-terminal) interfacial coupling free-energies. A bootstrap analysis is provided to estimate parameter uncertainty as well as correlations among fitted parameters. Rigorous statistical analysis is essential for interpreting fits using the complex models required for Ising analysis of repeat proteins, especially heteropolymeric repeat proteins. Programs described here are available athttps://github.com/barricklab-at-jhu/Ising_programs.

A collection of programs is presented to analyze the thermodynamics of folding of linear repeat proteins using a 1D Ising model to determine intrinsic folding and interfacial coupling free energies. Expressions for folding transitions are generated for a series of constructs with different repeat numbers and are globally fitted to transitions for these constructs. These programs are designed to analyze Ising parameters for capped homopolymeric consensus repeat constructs as well as heteropolymeric constructs that contain point substitutions, providing a rigorous framework for analysis of the effects of mutation on intrinsic and directional (i.e., N- vs. C-terminal) interfacial coupling free-energies. A bootstrap analysis is provided to estimate parameter uncertainty as well as correlations among fitted parameters. Rigorous statistical analysis is essential for interpreting fits using the complex models required for Ising analysis of repeat proteins, especially heteropolymeric repeat proteins. Programs described here are available at https://github.com/barricklab-at-jhu/Ising_programs.

Cryo-EM samples prepared using the traditional methods often suffer from too few particles, poor particle distribution, or strongly biased orientation, or damage from the air-water interface. Here we report that functionalization of graphene oxide (GO) coated grids with amino groups concentrates samples on the grid with improved distribution and orientation. By introducing a PEG spacer, particles are kept away from both the GO surface and the air-water interface, protecting them from potential denaturation.

DNA origami, a method for constructing nanoscale objects, relies on a long single strand of DNA to act as the ‘scaffold’ to template assembly of numerous short DNA oligonucleotide ‘staples’. The ability to generate custom scaffold sequences can greatly benefit DNA origami design processes. Custom scaffold sequences can provide better control of the overall size of the final object and better control of low-level structural details, such as locations of specific base pairs within an object. Filamentous bacteriophages and related phagemids can work well as sources of custom scaffold DNA. However, scaffolds derived from phages require inclusion of multi-kilobase DNA sequences in order to grow in host bacteria, and those sequences cannot be altered or removed. These fixed-sequence regions constrain the design possibilities of DNA origami. Here, we report the construction of a novel phagemid, pScaf, to produce scaffolds that have a custom sequence with a much smaller fixed region of 393 bases. We used pScaf to generate new scaffolds ranging in size from 1512 to 10 080 bases and demonstrated their use in various DNA origami shapes and assemblies. We anticipate our pScaf phagemid will enhance development of the DNA origami method and its future applications.

In biomolecules, bifurcated H-bonds typically involve the interaction of two donor protons with the two lone pairs of oxygen. Here, we present direct NMR evidence for a bifurcated H-bonding arrangement involving nitrogen as the acceptor atom. Specifically, the H-bond network comprises the Nδ1 atom of histidine and both the backbone N-H and side-chain Oγ-H of threonine within the conserved TXXH motif of ankyrin repeat (AR) proteins. Identification of the H-bonding partners is achieved via solution NMR H-bond scalar coupling (HBC) and H/D isotope shift experiments. Quantitative determination of (2h)J(NN) HBCs supports that Thr N-H···Nδ1 His H-bonds within internal repeats are stronger (∼4 Hz) than in the solvent exposed C-terminal AR (∼2 Hz). In agreement, pKa values for the buried histidines bridging internal ARs are several units lower than those of the C-terminus. Quantum chemical calculations show that the relevant (2h)J and (1h)J couplings are dominated by the Fermi contact interaction. Finally, a Thr-to-Val replacement, which eliminates the Thr Oγ-H···Nδ1 His H-bond and decreases protein stability, results in a 25% increase in (2h)J(NN), attributed to optimization of the Val N-H···Nδ1 His H-bond. Overall, the results provide new insights into the H-bonding properties of histidine, a refined structural rationalization for the folding cooperativity of AR proteins, and a challenging benchmark for the calculation of HBCs.

SummaryCooperativity is a defining feature of protein folding, but its thermodynamic and structural origins are not completely understood. By constructing consensus ankyrin repeat protein arrays that have nearly identical sequences, we quantify cooperativity by resolving stability into intrinsic and interfacial components. Heteronuclear NMR and CD spectroscopy show that these constructs adopt ankyrin repeat structures. Applying a one-dimensional Ising model to a series of constructs chosen to maximize information content in unfolding transitions, we quantify stabilities of the terminal capping repeats, and resolve the effects of denaturant into intrinsic and interfacial components. Reversible thermal denaturation resolves interfacial and intrinsic free energies into enthalpic, entropic, and heat capacity terms. Intrinsic folding is entropically disfavored, whereas interfacial interaction is entropically favored and attends a decrease in heat capacity. These results suggest that helix formation and backbone ordering occurs upon intrinsic folding, whereas hydrophobic desolvation occurs upon interfacial interaction, contributing to cooperativity.

Hypertrophic cardiomyopathy is the most frequently occurring inherited cardiovascular disease, with a prevalence of more than one in 500 individuals worldwide. Genetically acquired dilated cardiomyopathy is a related disease that is less prevalent. Both are caused by mutations in the genes encoding the fundamental force-generating protein machinery of the cardiac muscle sarcomere, including human β-cardiac myosin, the motor protein that powers ventricular contraction. Despite numerous studies, most performed with non-human or non-cardiac myosin, there is no clear consensus about the mechanism of action of these mutations on the function of human β-cardiac myosin. We are using a recombinantly expressed human β-cardiac myosin motor domain along with conventional and new methodologies to characterize the forces and velocities of the mutant myosins compared with wild type. Our studies are extending beyond myosin interactions with pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin, the roles of regulatory light chain phosphorylation on the functions of the system, and the possible roles of myosin binding protein-C and titin, important regulatory components of both cardiac and skeletal muscles.

Hydrogen bonds (H-bonds) are generally weak (< 20 kJ/mol) but ubiquitous interactions that determine in large part the cooperative folding and self-assembly of biological macromolecules in aqueous solution. Although important, most H-bonds are inferred indirectly and modeled using theoretical idealized geometry, therefore leaving instances of bifurcated or strained H-bonds undetected. Here, we present direct solution NMR evidence for an unusual bifurcated H-bonding arrangement comprising threonine Og-H and N-H donor protons and histidine acceptor Nd1 within the conserved TXXH alpha-helix N-capping motif of consensus ankyrin repeat (AR) proteins.The threonine N-H⋯Nd1 histidine H-bonds were detected in the TXXH motif of three and four-repeat AR proteins via 2hJNN hydrogen bond scalar coupling (HBC) NMR correlation experiments. Comparison of the 2hJNN couplings and histidine pKa values demonstrated that the N-H⋯Nd1 H-bonds of internal AR (2hJNN ∼ 4 Hz, pKa < 3) were stronger than those of the solvent-exposed C-terminal AR (2hJNN ∼ 2 Hz, pKa ∼ 5.7). In addition to the N-H⋯Nd1 2hJNN couplings, we also observed distinct 1hJHN HBCs connecting the threonine Og-H and histidine Nd1 within the TXXH motif of internal ARs. Together, the 2hJNN and 1hJHN HBCs supported that a single histidine Nd1 acceptor had two H-bond donors. Accordingly, two-bond reciprocal isotope shifts were detected between the threonine N-H and Og-H protons in solvents with mixed H2O/D2O composition. Finally, a threonine to valine replacement, which eliminated the Og-H⋯Nd1 H-bond and resulted in a destabilized protein, lead to a relaxed valine N-H⋯Nd1 histidine interaction with enhanced 2hJNN (∼5.2 Hz) likely owing to an optimized bonding geometry. Overall, our results provide new insight into the H-bonding ability of histidine and a challenging benchmark for the calculation of HBCs and their relation to H-bond energetics.Supported by NSF grant MCB-1330488

The application of hydrostatic pressure generally leads to protein unfolding, implying, in accordance with Le Chatelier's principle, that the unfolded state has a smaller molar volume than the folded state. However, the origin of the volume change upon unfolding, ΔV(u), has yet to be determined. We have examined systematically the effects of protein size and sequence on the value of ΔV(u) using as a model system a series of deletion variants of the ankyrin repeat domain of the Notch receptor. The results provide strong evidence in support of the notion that the major contributing factor to pressure effects on proteins is their imperfect internal packing in the folded state. These packing defects appear to be specifically localized in the 3D structure, in contrast to the uniformly distributed effects of temperature and denaturants that depend upon hydration of exposed surface area upon unfolding. Given its local nature, the extent to which pressure globally affects protein structure can inform on the degree of cooperativity and long-range coupling intrinsic to the folded state. We also show that the energetics of the protein's conformations can significantly modulate their volumetric properties, providing further insight into protein stability.

The linear “Ising” model, which has been around for nearly a century, treats the behavior of linear arrays of repetitive, interacting subunits. Linear “repeat-proteins” have only been described in the last decade or so, and their folding energies have only been characterized very recently. Owing to their repetitive structures, linear repeat-proteins are particularly well suited for analysis by the nearest-neighbor Ising formalism. After briefly describing the historical origins and applications of the Ising model to biopolymers, and introducing repeat protein structure, this chapter will focus on the application of the linear Ising model to repeat proteins. When applied to homopolymers, the model can be represented and applied in a fairly simplified form. When applied to heteropolymers, where differences in energies among individual subunits (i.e. repeats) must be included, some (but not all) of this simplicity is lost. Derivations of the linear Ising model for both homopolymer and heteropolymer repeat-proteins will be presented. With the increased complexity required for analysis of heteropolymeric repeat proteins, the ability to resolve different energy terms from experimental data can be compromised. Thus, a simple matrix approach will be developed to help inform on the degree to which different thermodynamic parameters can be extracted from a particular set of unfolding curves. Finally, we will describe the application of these models to analyze repeat-protein folding equilibria, focusing on simplified repeat proteins based on “consensus” sequence information.

The linear “Ising” model, which has been around for nearly a century, treats the behavior of linear arrays of repetitive, interacting subunits. Linear “repeat-proteins” have only been described in the last decade or so, and their folding energies have only been characterized very recently. Owing to their repetitive structures, linear repeat-proteins are particularly well suited for analysis by the nearest-neighbor Ising formalism. After briefly describing the historical origins and applications of the Ising model to biopolymers, and introducing repeat protein structure, this chapter will focus on the application of the linear Ising model to repeat proteins. When applied to homopolymers, the model can be represented and applied in a fairly simplified form. When applied to heteropolymers, where differences in energies among individual subunits (i.e. repeats) must be included, some (but not all) of this simplicity is lost. Derivations of the linear Ising model for both homopolymer and heteropolymer repeat-proteins will be presented. With the increased complexity required for analysis of heteropolymeric repeat proteins, the ability to resolve different energy terms from experimental data can be compromised. Thus, a simple matrix approach will be developed to help inform on the degree to which different thermodynamic parameters can be extracted from a particular set of unfolding curves. Finally, we will describe the application of these models to analyze repeat-protein folding equilibria, focusing on simplified repeat proteins based on “consensus” sequence information.

Repeat proteins have been popular model systems in protein folding research due to their modular architecture. Although each repeat has the same fold, because of sequence variation, the folding energy is not distributed evenly among the repeats. Therefore, it is challenging to quantify the repeat-repeat interactions and the intrinsic folding energies of every repeat. To overcome this difficulty, we designed a consensus ankyrin repeat(CANK), which is brought together to make arrays of identical repeats. By solution NMR, we solved the structure of our three repeat CANK, NRC, and verify that NRC adopts the canonical Ankyrin repeat protein fold. Our SAXS measurements show that CANKs in different length arrays also adopt the same fold. By fitting unfolding transitions of a limited number of constructs to Ising model, we found that the individual repeats have low intrinsic stability, but the interfacial interactions are highly stabilizing. We also extended our Ising formalism to include the temperature dependence of unfolding free energy and we quantified the enthalpic and entropic contribution to intrinsic and interfacial stabilities, as well as, the uneven distribution of heat capacity of unfolding into the intrinsic and interfacial components. Our results suggest that intrinsic folding resembles secondary structure formation(loss of conformational entropy) and interfacial folding involves hydrophobic desolvation(entropy increase and negative heat capacity change upon folding). To separate out the contribution of long range electrostatics to intrinsic and interfacial stability, we measured the salt dependence of CANK stabilities. Our results show that NaCl stabilizes the interfaces and has marginal destabilizing effect on intrinsic stability. Currently we are trying to identify the charge-charge interactions responsible for the high salt dependence of interfacial stability by NMR techniques and single point substitutions.

Although progress has been made to determine the native fold of a polypeptide from its primary structure, the diversity of pathways that connect the unfolded and folded states has not been adequately explored. Theoretical and computational studies predict that proteins fold through parallel pathways on funneled energy landscapes, although experimental detection of pathway diversity has been challenging. Here, we exploit the high translational symmetry and the direct length variation afforded by linear repeat proteins to directly detect folding through parallel pathways. By comparing folding rates of consensus ankyrin repeat proteins (CARPs), we find a clear increase in folding rates with increasing size and repeat number, although the size of the transition states (estimated from denaturant sensitivity) remains unchanged. The increase in folding rate with chain length, as opposed to a decrease expected from typical models for globular proteins, is a clear demonstration of parallel pathways. This conclusion is not dependent on extensive curve-fitting or structural perturbation of protein structure. By globally fitting a simple parallel-Ising pathway model, we have directly measured nucleation and propagation rates in protein folding, and have quantified the fluxes along each path, providing a detailed energy landscape for folding. This finding of parallel pathways differs from results from kinetic studies of repeat-proteins composed of sequence-variable repeats, where modest repeat-to-repeat energy variation coalesces folding into a single, dominant channel. Thus, for globular proteins, which have much higher variation in local structure and topology, parallel pathways are expected to be the exception rather than the rule.

Pressure effects of protein conformational transitions, which are manifest via the associated changes in molar volume, remain rather poorly understood, and regrettably so given that they are clearly linked to changes in the magnitude and type of hydration that accompany such transitions. Given that the role of solvent in protein energetics and structural dynamics remains one of the key questions in the field of protein folding, a better understanding of the physical basis for the volume changes that accompany folding reactions could provide direct means to quantify this differential solvation. We have recently begun high pressure studies on the folding of the ankyrin repeat domain of the protein Notch bearing 7 ankyrin repeats (Nank7) and a series of smaller constructs differing is the number of repeats, and/or their sequences. We reasoned that such a system would provide a means of incrementally testing the role of the size of the hydrophobic core, and the importance of the specific amino acid sequence in determining the volume change upon unfolding, ΔVu. We present here a complete P-T equilibrium and p-jump kinetic study of the full-length Nank7 construct, as well as on a number of the smaller constructs. We find that the volume change of the full-length construct depends strongly upon temperature, and that the expansivity of the transition state ensemble is similar to that of the unfolded state, while its molar volume is closer to that of the folded state. Preliminary results on two very small constructs indicate that the total volume change is a linear combination of the volume change associated with the unfolding of each individual repeat.

Hypertrophic (HCM) and dilated (DCM) cardiomyopathies are important causes of heart failure, arrhythmia, and sudden cardiac death. HCM is the most common heritable cardiovascular disorder, affecting 1 in 500 individuals, and is caused primarily by mutations that alter proteins of the cardiac sarcomere. Sarcomeric protein mutations are an increasingly recognized cause of familial DCM as well. HCM is associated with severe thickening of the left ventricular wall, preserved/increased systolic (contractile) and impaired diastolic (relaxation) function of the heart. DCM hearts have dilated left ventricular chambers and suffer from inadequate systolic activity. More than 300 point mutations in beta-cardiac myosin are associated with HCM or DCM. It is believed that HCM and DCM mutations increase and decrease respectively the power output of cardiac myosin which leads to a cascade of downstream signaling that gradually leads to the disease phenotype. To test this hypothesis it is essential to measure the power output of all HCM/DCM mutants in a high throughput manner. To quantitatively measure the load dependent myosin power output, we first improved the loaded in vitro motility assay (LIMA) by replacing load generating molecule alpha-actinin with alpha-catenin, which was generously provided by James Nelson at Stanford University. For accurate-and-fast data analysis, we developed a software called FAST that runs over parallel CPUs. Our initial results indicate that two HCM mutants (R719W, R403Q) have higher power output than the wildtype cardiac myosin. FAST-LIMA will be an essential tool for screening drugs that revert the effects of HCM/DCM mutations on the power output of beta-cardiac myosin.

Understanding the intricate relationships between protein sequence, structure, and stability remains a challenge. One major obstacle in understanding these relationships is the identification of interactions that couple elements of structure; these interactions are not readily apparent from structure. Recently, the application of nearest-neighbor models to repeat protein folding have begun to provide insights into how these systems distribute their energy within, and between units of protein structure1,2,3. Consensus ankyrin and 34-residue cTPR protein (aka c34PR) folding can be described using two terms: the intrinsic energy of individual repeats (ΔGi), and the interfacial energy between adjacent repeats (ΔGi,i+1).Here, we extend this approach in two ways using two experimental TPR-like systems. First, we dissect the whole-repeat nearest-neighbor model by representing (and resolving) the subunits of the nearest-neighbor model into each of the two helices (“A” and “B”) of each repeat. This extended Ising analysis allows us to quantify coupling energies within and between repeats, as well as local stability differences of the A and B repeat helices. Second, we explore a new, longer class of TPR-like proteins with 42 residues per repeat (42PRs, as opposed to 34 residues for the founding TPR sequence motif), to determine how variation in helix length and interface structure affects intrinsic and coupling energy.By varying the length of constructs in half-repeat increments, we find energetic heterogeneity at both the intrinsic and interfacial level. Although stabilities are more homogeneously distributed in the c34PR series (ΔGAB and ΔGBA as well as ΔGA and ΔGB are nearly isoenergetic), the distribution is more heterogeneous within 42PRs, with a greater energetic separation between intrinsic and interfacial energies. This leads to greater folding cooperativity within the 42PR series.[1] Wetzel and Pluckthun, JMB[2] Aksel, Structure[3] Kajandar et al, JACS