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A grand challenge in biosensor design is to develop a single molecule, fluorescent protein-based platform that can be easily adapted to recognize targets of choice. Conceptually, this can be achieved by fusing a small, antibody-like binding domain to a fluorescent protein in such a way that target binding activates fluorescence. Although this design is simple to envision, its execution is not obvious. Here, we created a family of adaptable, turn-on monobody (ATOM) biosensors consisting of a monobody, circularly permuted at one of two positions, inserted into a fluorescent protein at one of three surface loops. Multiplexed imaging of live human cells co-expressing cyan, yellow, and red ATOM sensors detected the biosensor targets (WDR5, SH2, and hRAS proteins) that were localized to the nucleus, cytoplasm, and plasma membrane, respectively, with high specificity. ER- and mitochondria-localized ATOM sensors also detected ligands that were targeted to those organelles. Fluorescence activation involved ligand-dependent chromophore maturation with fluorescence turn-on ratios of >20-fold in cells and up to 100-foldin vitro. The sensing mechanism was validated with three arbitrarily chosen monobodies inserted into jellyfish as well as anemone lineages of fluorescent proteins, suggesting that ATOM sensors with different binding specificities and additional colors can be generated relatively quickly.

Protein conformational switches are widely used in biosensing. They are often composed of an input domain (which binds a target ligand) fused to an output domain (which generates an optical readout). A central challenge in designing such switches is to develop mechanisms for coupling the input and output signals

Protein detection and biomarker profiling have implications in basic research and molecular diagnostics. Substantial progress has been made in protein analytics using nanopores and the resistive-pulse technique. Yet, a long-standing challenge is implementing specific binding interfaces for detecting proteins without the steric hindrance of the pore interior. To overcome this technological difficulty, we formulate a new class of sensing elements made of a programmable antibody-mimetic binder fused to a monomeric protein nanopore. This way, such a modular design significantly expands the utility of nanopore sensors to numerous proteins while preserving their architecture, specificity, and sensitivity. We prove the power of this approach by developing and validating nanopore sensors for protein analytes that drastically vary in size, charge, and structural complexity. These analytes produce unique electrical signatures that depend on their identity and quantity and the binder-analyte assembly at the nanopore tip. From a practical point of view, our sensors unambiguously probe protein recognition events without using any additional exogenous tag. The outcomes of this work will impact biomedical diagnostics by providing a fundamental basis and tools for protein biomarker detection in biofluids.

Protein detection and biomarker profiling have wide-ranging implications in many areas of basic research and molecular diagnostics. Substantial progress has been made in protein analytics using nanopores and the resistive-pulse technique. Yet, a long-standing challenge is implementing specific binding interfaces for detecting proteins without the steric hindrance of the pore interior. To overcome this technological difficulty, we formulate a new class of sensing elements made of a programmable antibody-mimetic binder fused to a monomeric protein nanopore. This way, such a modular design significantly expands the utility of nanopore sensors to numerous proteins while preserving their architecture, specificity, and sensitivity. We prove the power of this approach by developing and validating nanopore sensors for protein analytes that drastically vary in size, charge, and structural complexity. These analytes produce unique electrical signatures that depend on their identity and quantity and the binder-analyte assembly at the nanopore tip. From a practical point of view, our sensors unambiguously probe protein recognition events without the necessity of using any additional exogenous tag. The outcomes of this work will impact biomedical diagnostics by providing a fundamental basis and tools for protein biomarker detection in biofluids.

Switchable proteins are capable of changing conformations from inactive (OFF) to active (ON) forms in response to inputs such as ligand binding, pH or temperature change, or light absorption. A particularly powerful class of protein switches, exemplified by the Cas nucleases of CRISPR systems, are activated by binding of specific DNA or RNA sequences. The mechanism by which oligonucleotide binding regulates biological activity is complex and highly specialized in the case of Cas enzymes, but recent advancements in protein and DNA engineering have made it possible to introduce this mode of control into other enzymes. This chapter highlights recent examples of protein switches that combine these two fields of engineering for the purpose of creating biosensors that detect pathogen and other genomic sequences. One protein engineering method-alternate frame folding-has the potential to convert many proteins into ligand-activated switches by inserting a binding protein (input domain) into an enzyme (output domain). The steps for doing so are illustrated using GCN4 as a DNA recognition domain and nanoluciferase as a luminescent reporter that changes color as a result of DNA binding. DNA engineering protocols are included for creating DNA tools (de novo designed hairpins and modified aptamers), that enable the biosensor to be activated by arbitrary DNA/RNA sequences and small molecules/proteins, respectively. These methodologies can be applied to other proteins to gain control of their functions by DNA binding.

Protein-based fluorescent biosensors are powerful tools for analyte recognition in vitro and in cells. Numerous proteinaceous binding scaffolds have been developed that recognize ligands with affinity and specificity comparable to those of conventional antibodies, but are smaller, readily overexpressed, and more amenable to engineering. Like antibodies, these binding domains are useful as recognition modules in protein switches and biosensors, but they are not capable of reporting on the binding event by themselves. Here, we engineer a small binding scaffold—a consensus-designed fibronectin 3 monobody—such that it undergoes a conformational change upon ligand binding. This change is detected by Förster resonance energy transfer using chemical dyes or cyan and yellow fluorescent proteins as donor/acceptor groups. By grafting substrate recognition residues from different monobodies onto this scaffold, we create fluorescent biosensors for c-Abl Src homology 2 (SH2) domain, WD40-repeat protein 5 (WDR5), small ubiquitin-like modifier-1 (SUMO), and h-Ras. The biosensors bind their cognate ligands reversibly, with affinities consistent with those of the parent monobodies, and with half times of seconds to minutes. This design serves as generalizable platform for creating a genetically-encoded, ratiometric biosensors by swapping binding residues from known monobodies, with minimal modification.

Here, we describe the use of artificial intelligence to identify novel agonists of the SH2-containing 5' inositol phosphatase 1 (SHIP1). One of the compounds, K306, represents the most potent agonist identified to date. We find that K306 exhibits selectivity for SHIP1 vs. the paralog enzyme SHIP2, and this activation does not require the C2 domain of SHIP1 which other known SHIP1 agonists require. Thus, K306 represents a new class of SHIP1 agonists with a novel mode of agonism. Importantly, we find that K306 can suppress induction of inflammatory cytokines and iNOS in macrophages or microglia, but not by their SHIP1-deficient counterparts. K306 also reduces TNF-α production

In the cell, the thermodynamic stability of a protein - and hence its biological activity - can change dramatically as a result of perturbations in its amino acid sequence and the concentration of stabilizing ligands. This interplay is particularly evident in zinc-binding transcription factors such as the p53 tumor suppressor, whose DNA-binding activity can critically depend on levels of intracellular zinc as well as point mutations that alter either metal binding or folding stability. Separate protocols exist for determining a protein's metal affinity and its folding free energy. These properties, however, are intimately connected, and a technique is needed to integrate these measurements. Our protocols employ common non-fluorescent and fluorescent zinc chelators to control and report on free Zn2+ concentration, respectively, combined with biophysical assays of full-length human p53 and its DNA-binding domain. Fitting the data to equations that contain stability and metal-binding terms results in a more complete picture of how metal-dependent proteins can lose and gain DNA-binding function in a range of physiological conditions. Graphic abstract: Figure 1.Raising intracellular zinc can restore tumor-suppressing function to p53 that has been unfolded by missense mutation or cellular conditions.

Epigallocatechin gallate (EGCG) from green tea can induce apoptosis in cancerous cells, but the underlying molecular mechanisms remain poorly understood. Using SPR and NMR, here we report a direct, μM interaction between EGCG and the tumor suppressor p53 (KD = 1.6 ± 1.4 μM), with the disordered N-terminal domain (NTD) identified as the major binding site (KD = 4 ± 2 μM). Large scale atomistic simulations (>100 μs), SAXS and AUC demonstrate that EGCG-NTD interaction is dynamic and EGCG causes the emergence of a subpopulation of compact bound conformations. The EGCG-p53 interaction disrupts p53 interaction with its regulatory E3 ligase MDM2 and inhibits ubiquitination of p53 by MDM2 in an in vitro ubiquitination assay, likely stabilizing p53 for anti-tumor activity. Our work provides insights into the mechanisms for EGCG’s anticancer activity and identifies p53 NTD as a target for cancer drug discovery through dynamic interactions with small molecules., Epigallocatechin gallate (EGCG) is a catechin flavonoid which induces apoptosis in cancerous cells, but the underlying molecular mechanisms remain poorly understood. Here authors use an interdisciplinary approach to show a direct interaction between EGCG and the tumor suppressor p53 and demonstrate that EGCG inhibits ubiquitination of p53 by MDM2.

Aggregation of the circulating protein leukocyte-cell-derived chemotaxin 2 (LECT2) causes amyloidosis of LECT2 (ALECT2), one of the most prevalent forms of systemic amyloidosis affecting the kidney and liver. The I40V mutation is thought to be necessary but not sufficient for ALECT2, with a second, as-yet undetermined condition being required for the disease. EM, X-ray diffraction, NMR, and fluorescence experiments demonstrate that LECT2 forms amyloid fibrils in vitro in the absence of other proteins. Removal of LECT2’s single bound Zn2+ appears to be obligatory for fibril formation. Zinc-binding affinity is strongly dependent on pH: 9–13 % of LECT2 is calculated to exist in the zinc-free state over the normal pH range of blood, with this fraction rising to 80 % at pH 6.5. The I40V mutation does not alter zinc-binding affinity or kinetics but destabilizes the zinc-free conformation. These results suggest a mechanism in which loss of zinc together with the I40V mutation leads to ALECT2.

Missense mutations in the p53 DNA-binding domain (DBD) contribute to half of new cancer cases annually. Here we present a thermodynamic model that quantifies and links the major pathways by which mutations inactivate p53. We find that DBD possesses two unusual properties—one of the highest zinc affinities of any eukaryotic protein and extreme instability in the absence of zinc—which are predicted to poise p53 on the cusp of folding/unfolding in the cell, with a major determinant being available zinc concentration. We analyze the 20 most common tumorigenic p53 mutations and find that 80% impair zinc affinity, thermodynamic stability, or both. Biophysical, cell-based, and murine xenograft experiments demonstrate that a synthetic zinc metallochaperone rescues not only mutations that decrease zinc affinity, but also mutations that destabilize DBD without impairing zinc binding. The results suggest that zinc metallochaperones have the capability to treat 120,500 patients annually in the U.S.

In the past decade, significant progress has been made in the development of new protein nanopores. Despite these advancements, there is a pressing need for the creation of nanopores equipped with relatively large functional groups for the sampling of biomolecular events on their extramembranous side. Here, we designed, produced, and analyzed protein nanopores encompassing a robust truncation of a monomeric β-barrel membrane protein. An exogenous stably folded protein was anchored within the aqueous phase via a flexible peptide tether of varying length. We have extensively examined the pore-forming properties of these modular protein nanopores using protein engineering and single-molecule electrophysiology. This study revealed distinctions in the nanopore conductance and current fluctuations that arose from tethering the exogenous protein to either the N terminus or the C terminus. Remarkably, these nanopores insert into a planar lipid membrane with one specific conductance among a set of three substate conductance values. Moreover, we demonstrate that the occurrence probabilities of these insertion substates depend on the length of the peptide tether, the orientation of the exogenous protein with respect to the nanopore opening, and the molecular mass of tethered protein. In addition, the three conductance values remain unaltered by major changes in the composition of modular nanopores. The outcomes of this work serve as a platform for further developments in areas of protein engineering of transmembrane pores and biosensor technology.

Missense mutations in the DNA binding domain (DBD) of the p53 tumor suppressor contribute to approximately half of new cancer cases each year worldwide. A primary goal in cancer therapy is to develop drugs that rescue the transcription function of mutant p53. Here we present a thermodynamic model that quantifies and links the major pathways by which mutations inactivate p53. The model is constructed by measuring folding free energies, zinc dissociation constants, and DNA dissociation constants of 20 of the most common DBD mutations in the p53 database. We report here that DBD possesses two unusual properties——one of the highest zinc binding affinities of any eukaryotic protein and extreme instability in the absence of zinc—which are predicted to cause p53 to be poised on the edge of folding/unfolding in the cell, with a major determinant being the concentration of available zinc. Eighty percent of the mutations examined impair either thermodynamic stability, zinc binding affinity, or both. Using a combination of biophysical experiments, cell based assays, and murine cancer models, we demonstrate for the first time that a synthetic zinc metallochaperone not only rescues mutants with decreased zinc affinities, but also mutants that destabilize DBD without impairing zinc binding. The latter is a broad class of p53 mutants of which only one member (Y220C) has been successfully targeted by small molecules. The results suggest that zinc metallochaperones have the capability to treat 120,500 patients per year in the U.S.SUMMARYRestoring tumor suppressing function to mutant p53 has the capability of treating millions of new cancer patients worldwide each year. An important step toward this goal is to categorize the spectrum of mutations based on how they inactivate p53. This study finds that the majority of the most common tumorigenic mutations compromise p53’s thermodynamic stability or its interaction with zinc, and demonstrates for the first time that members of both classes can be reactivated in cells by synthetic zinc metallochaperones. These results serve to stratify patients for potential zinc metallochaperone therapy.

The design of protein conformational switches—or proteins that change conformations in response to a signal such as ligand binding—has great potential for developing novel biosensors, diagnostic tools, and therapeutic agents. Among the defining properties of such switches, the response time has been the most challenging to optimize. Here we apply a computational design strategy in synergistic combination with biophysical experiments to rationally improve the response time of an engineered protein-based Ca2+-sensor in which the switching process occurs via mutually exclusive folding of two alternate frames. Notably, our strategy identifies mutations that increase switching rates by as much as 32-fold, achieving response times on the order of fast physiological Ca2+ fluctuations. Our computational design strategy is general and may aid in optimizing the kinetics of other protein conformational switches., The rational optimization of response times of protein conformational switches is a major challenge for biomolecular switch design. Here the authors present a generally applicable computational design strategy that in combination with biophysical experiments can improve response times using a Ca2+-sensor as an example.

Disrupting a protein’s sequence by cleavage or insertion of a hinge domain forms the basis for protein engineering tools, including fragment complementation, circular permutation, and domain swapping. Despite the utility of these designs, their widespread implementation has been limited by the difficulty in choosing where to interrupt the protein sequence: the resulting fragments often aggregate or fail to reassemble. Here, we show that an optimal site exists within ribose binding protein (RBP) that, when disrupted, results in the most efficient formation of fragment-complemented and domain-swapped species. Cleaving RBP at this site also produces a highly stable, cooperatively folded circular permutant. This hot-spot site was identified by an experimental approach involving selection among competing folds. We find that efficiency in the case of RBP is determined by kinetic factors (survival of the first) rather than thermodynamics (survival of the fittest). Together with emerging computational tools, this limited data set defines a pathway for designing robust platforms for molecular switches and biosensors based on the aforementioned protein modifications.

Alternate frame folding (AFF) and protein/fragment exchange (FREX) are related technologies for engineering allosteric conformational changes into proteins that have no pre-existing allosteric properties. One of their chief purposes is to turn an ordinary protein into a biomolecular switch capable of transforming an input event into an optical or functional readout. Here, we present a guide for converting an arbitrary binding protein into a fluorescent biosensor with Förster resonance energy transfer output. Because the AFF and FREX mechanisms are founded on general principles of protein structure and stability rather than a property that is idiosyncratic to the target protein, the basic design steps—choice of permutation/cleavage sites, molecular biology, and construct optimization—remain the same for any target protein. We highlight effective strategies as well as common pitfalls based on our experience with multiple AFF and FREX constructs.

Domain-swapped proteins are found throughout nature and play important roles in biology, e.g. cell-cell adhesion in the case of cadherins, as well as in some deposition disorders such as human prion disease. Domain-swapped proteins form dimers and oligomers by reciprocal exchange of polypeptides. The structure of the swapped oligomer is identical to that of the non-swapped monomer except at the site of strand exchange (hinge region). Here we show a general method for designing functional polymers through engineered domain swapping. Domain swapping is induced in a functional protein by inserting a small, stable protein (“lever”) with a long distance between its N- and C-termini into a surface loop of the functional host protein. The lever domain applies conformational stress to the host domain, ripping it apart at the point of insertion. Conformational stress is relieved when two or more host-lever fusion proteins refold intermolecularly via domain swap of their host domains. The function of the host domain is controlled by creating two constructs: one with a mutation disabling the function of the host introduced N-terminal to the lever insertion point, and one with another such mutation introduced C-terminal to the insertion point. The monomeric proteins are in the nonfunctional OFF state, but when they domain swap the wild-type sequence of the host is restored, resulting in the ON state. We have developed a family of host-lever constructs (RU series) in which ubiquitin (lever) is inserted into different surface loops of ribose binding protein (RBP; host). Here we use crosslinking, functional, and NMR structural experiments to demonstrate the successful switching of ribose binding function through induced domain swapping and have determined the preferred hinge region for swapping of RBP.

Protein conformational switches alter their shape upon receiving an input signal, such as ligand binding, chemical modification, or change in environment. The apparent simplicity of this transformation--which can be carried out by a molecule as small as a thousand atoms or so--belies its critical importance to the life of the cell as well as its capacity for engineering by humans. In the realm of molecular switches, proteins are unique because they are capable of performing a variety of biological functions. Switchable proteins are therefore of high interest to the fields of biology, biotechnology, and medicine. These molecules are beginning to be exploited as the core machinery behind a new generation of biosensors, functionally regulated enzymes, and "smart" biomaterials that react to their surroundings. As inspirations for these designs, researchers continue to analyze existing examples of allosteric proteins. Recent years have also witnessed the development of new methodologies for introducing conformational change into proteins that previously had none. Herein we review examples of both natural and engineered protein switches in the context of four basic modes of conformational change: rigid-body domain movement, limited structural rearrangement, global fold switching, and folding-unfolding. Our purpose is to highlight examples that can potentially serve as platforms for the design of custom switches. Accordingly, we focus on inducible conformational changes that are substantial enough to produce a functional response (e.g., in a second protein to which it is fused), yet are relatively simple, structurally well-characterized, and amenable to protein engineering efforts.

Many proteins are built from structurally and functionally distinct and domains. A major goal is to understand how conformational change transmits information between domains in order to achieve biological activity. A two-domain, bi-functional fusion protein has been designed so that the mechanical stress imposed by the folded structure of one subunit causes the other subunit to unfold, and vice versa. The construct consists of ubiquitin inserted into a surface loop of barnase. The distance between the amino and carboxyl ends of ubiquitin is much greater than the distance between the termini of the barnase loop. This topological constraint causes the two domains to engage in a thermodynamic tug-of-war in which only one can exist in its folded state at any given time. This conformational equilibrium, which is cooperative, reversible, and controllable by ligand binding, serves as a model for the coupled binding and folding mechanism widely used to mediate protein–protein interactions and cellular signaling processes. The position of the equilibrium can be adjusted by temperature or ligand binding and is monitored in vivo by cell death. This design forms the basis for a new class of cytotoxic proteins that can be activated by cell-specific effector molecules, and can thus target particular cell types for destruction.

The chemical shifts of the C(epsilon1) and C(delta2) protons of His-240 from the 109 kDa Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PD) were assigned by comparing 1H and 13C spectra of the wild-type and mutant G6PDs containing the His-240 to asparagine mutation (H240N). Unambiguous assignment of the His-240 1H(epsilon1) resonance was obtained from comparing 13C-1H heteronuclear multiple quantum coherence NMR spectra of wild-type and H240N G6PDs that were selectively labeled with 13C(epsilon1) histidine. The results from NOESY experiments with wild-type and H240N variants were consistent with these assignments and the three-dimensional structure of G6PD. pH titrations show that His-240 has a pK(a) of 6.4. This value is, within experimental error, identical to the value of 6.3 derived from the pH dependence of kcat [Viola, R. E. (1984) Arch. Biochem. Biophys. 228, 415-424], suggesting that the pK(a) of His-240 is unperturbed in the apoenzyme despite being part of a His-Asp catalytic dyad. The results obtained for this 109 kDa enzyme indicate that 1H NMR spectroscopy in combination with heteronuclear methods can be a useful tool for functional analysis of large proteins.

We have compared 70-kDa heat shock cognate protein (Hsc70) isolated from bovine brain with recombinant wild type protein and mutant E543K protein (previously studied as wild type in our laboratory). Wild type bovine and recombinant protein differ by posttranslational modification of lysine 561 but interact similarly with a short peptide (fluorescein-labeled FYQLALT) and with denatured staphylococcal nuclease-(Delta135-149). Mutation E543K results in 4. 5-fold faster release of peptide and lower stability of complexes with staphylococcal nuclease-(Delta135-149). ATP hydrolysis rates of the wild type proteins are enhanced 6-10-fold by the addition of peptide. The E543K mutant has a peptide-stimulated hydrolytic rate similar to that of wild type protein but a higher unstimulated rate, yielding a mere 2-fold enhancement. All three versions of Hsc70 possess similar ATP-dependent conformational shifts, and all show potassium ion dependence. These data support the following model: (i) in the presence of K+, Mg2+, and ATP, the peptide binding domain inhibits the ATPase; (ii) binding of peptide relieves this inhibition; and (iii) the E543K mutation significantly attenuates the inhibition by the peptide binding domain and destabilizes Hsc70-peptide complexes.

Alternate frame folding (AFF) is a protein engineering methodology the purpose of which is to convert an ordinary binding protein into a molecular switch. The AFF modification entails duplicating an amino- or carboxy-terminal segment of the protein and appending it to the opposite end of the molecule. This duplication allows the protein to interconvert, in a ligand-dependent fashion, between two mutually exclusive native folds: the wild-type structure and a circularly permuted form. The fold shift can be detected by placement of extrinsic fluorophores at sites sensitive to the engineered conformational change. Here, we apply the AFF mechanism to create several ribose-sensing proteins derived from Thermoanaerobacter tengcongensis ribose binding protein. Our purpose is to systematically explore the parameters of the AFF design. These considerations include the site of circular permutation, the length and location of the duplicated segment, thermodynamic and kinetic optimization of the switching mechanism, and placement of extrinsic fluorophores. Three of the four AFF variants created here undergo the expected conformational shift and exhibit a ribose-dependent fluorescence change. The fourth construct fails to switch folds upon addition of ribose, likely because the circularly permuted form folds much more slowly than the nonpermuted form. This disparity apparently introduces a kinetic barrier that partitions the refolding molecules to the nonpermuted structure. The results of this study serve as a guideline for applying the AFF modification to other proteins of biomedical, diagnostic, and industrial interest.

Angiogenin (ANG) promotes the formation of blood vessels in animals. This hormone is a small, monomeric protein that is homologous to bovine pancreatic ribonuclease A (RNase). ANG is a poor ribonuclease but its ribonucleolytic activity is essential for its angiogenic activity. RNase is not angiogenic. A hybrid protein was produced in which 13 residues of a divergent surface loop of ANG were substituted for the analogous 15 residues of RNase. The value of k/K for the cleavage of uridylyl(3′5′)adenosine by this hybrid protein was 20-fold less than that of RNase but 105-fold greater than that of ANG. The thermal stability of the hybrid protein was also less than that of RNase. Nevertheless, the RNase/ANG hybrid protein promotes angiogenesis in mice at least as extensively as does authentic ANG. Thus we present a protein endowed with a noncognate biological activity simply by replacing a single element of secondary structure. In addition, a 13-residue peptide corresponding to the surface loop of ANG inhibits endogenous angiogenesis in mice. These results support a model in which both a surface loop and a catalytic site are necessary for the promotion of blood vessel formation by ANG or RNase. The dissection of structure/function elements in ANG reveals a unique opportunity to develop new molecules that modulate neovascularization.

Steady-state kinetic, pre-steady-state kinetic, and equilibrium binding measurements have been applied to determine the rate constants of individual steps of the ATPase cycle for the recombinant bovine 70 kDa heat shock cognate protein and its amino-terminal 44 kDa ATPase fragment. At 25 degrees C, pH 7.0, in the presence of 75 mM KCl and 4.5 mM Mg2+, the measured association rate constants for MgATP approximately hsc70 and MgADP approximately hsc70 are (2.7 +/- 0.5) x 10(5) and (4.1 +/- 0.5) x 10(5) M-1 s-1, respectively, while the dissociation rate constants are 0.0114 (+/- 0.0002) and 0.0288 (+/- 0.0018) s-1, respectively. MgATP (Kd = 0.042 microM) therefore binds to hsc70 more tightly than MgADP (Kd = 0.11 microM). ADP release is inhibited by inorganic phosphate (Pi), suggesting that product dissociation is ordered with Pi released first and ADP second. The rate of chemical hydrolysis of ATP is 0.0030 (+/- 0.0003) s-1 for hsc70 and 0.0135 (+/- 0.0033) s-1 for the 44 kDa fragment. The rate of Pi release is 0.0038 (+/- 0.0010) s-1 for hsc70 and 0.0051 (+/- 0.0006) s-1 for the 44 kDa fragment. For the 44 kDa fragment, Pi release is the slowest step in the ATPase cycle, while for hsc70, Pi release and chemical hydrolysis of MgATP have similar rates; in both cases, ADP release is a relatively rapid step in the ATPase cycle.

Domain swapping is a mechanism for forming protein dimers and oligomers with high specificity. It is distinct from other forms of oligomerization in that the binding interface is formed by reciprocal exchange of polypeptide segments. Swapping plays a physiological role in protein-protein recognition and it can also potentially be exploited as a mechanism for controlled self assembly. Here, we demonstrate that domain-swapped interfaces can be engineered by inserting one protein into a surface loop of another protein. The key to facilitating a domain swap is to destabilize the protein when it is monomeric but not when it is oligomeric. We achieve this condition by employing the ‘mutually exclusive folding’ design to apply conformational stress to the monomeric state. Ubiquitin is inserted into one of six surface loops of barnase. The 38 Å amino-to-carboxy terminal distance of ubiquitin stresses the barnase monomer, causing it to split at the point of insertion. The 2.2 Å X-ray structure of one insertion variant reveals that strain is relieved by intermolecular folding with an identically-unfolded barnase domain, resulting in a domain-swapped polymer. All six constructs oligomerize suggesting that inserting ubiquitin into each surface loop of barnase results in a similar domain-swapping event. Binding affinity can be tuned by varying the length of the peptide linkers used to join the two proteins, which modulates the extent of stress. Engineered, swapped proteins have the potential to be used to fabricate ‘smart’ biomaterials, or as binding modules from which to assemble heterologous, multi-subunit protein complexes.

The objective of this study is to quantify the contributions of cations, anions and water to stability and specificity of the interaction of lac repressor (lac R) protein with the strong-binding symmetric lac operator (Osym) DNA site. To this end, binding constants Kobs and their power dependences on univalent salt (MX) concentration (SKobs = d log Kobs/d log[MX]) have been determined for the interactions of lac R with Osym operator and with non-operator DNA using filter binding and DNA cellulose chromatography, respectively. For both specific and non-specific binding of lac R, Kobs at fixed salt concentration [KX] increases when chloride (Cl-) is replaced by the physiological anion glutamate (Glu-). At 0.25 M-KX, the increase in Kobs for Osym is observed to be approximately 40-fold, whereas for non-operator DNA the increase in Kobs is estimated by extrapolation to be approximately 300-fold. For non-operator DNA, SKobsRD is independent of salt concentration within experimental uncertainty, and is similar in KCl (SKobs,RDKCl = -9.8(+/- 1.0) between 0.13 M and 0.18 M-KCl) and KGlu (SKobs,RDKGlu = -9.3(+/- 0.7) between 0.23 M and 0.36 M-KGlu). For Osym DNA, SKobsRO varies significantly with the nature of the anion, and, at least in KGlu appears to decrease in magnitude with increasing [KGlu]. Average magnitudes of SKobsRO are less than SKobsRD, and, for specific binding decrease in the order [SKobsRO,KCl[>[SKobsRO,KAc[>[SKobsRO,KGlu[ . Neither KobsRO nor SKobsRO is affected by the choice of univalent cation M+ (Na+, K+, NH4+, or mixtures thereof, all as the chloride salt), and SKobsRO is independent of [MCl] in the range examined (0.125 to 0.3 M). This behavior of SKobsRO is consistent with that expected for a binding process with a large contribution from the polyelectrolyte effect. However, the lack of an effect of the nature of the cation on the magnitude of KobsRO at a fixed [MX] is somewhat unexpected, in view of the order of preference of cations for the immediate vicinity of DNA (NH4+ > K+ > Na+) observed by 23Na nuclear magnetic resonance. For both specific and non-specific binding, the large stoichiometry of cation release from the DNA polyelectrolyte is the dominant contribution to SKobs. To interpret these data, we propose that Glu- is an inert anion, whereas Ac- and Cl- compete with DNA phosphate groups in binding to lac repressor. A thermodynamic estimate of the minimum stoichiometry of water release from lac repressor and Osym operator (210(+/- 30) H2O) is determined from analysis of the apparently significant reduction in [SKobsRO,KGlu[ with increasing [KGlu] in the range 0.25 to 0.9 M. According to this analysis, SKobs values of specific and non-specific binding in KGlu differ primarily because of the release of water in specific binding. In KAc and KCl, we deduce that anion competition affects Kobs and SKobs to an extent which differs for different anions and for the different binding modes.

A regulatory mechanism is introduced whereupon the catalytic activity of a given enzyme is controlled by ligand binding to a receptor domain of choice. A small enzyme (barnase) and a ligand-binding polypeptide (GCN4) are fused so that a simple topological constraint prevents them from existing simultaneously in their folded states. The two domains consequently engage in a thermodynamic tug-of-war in which the more stable domain forces the less stable domain to unfold. In the absence of ligand, the barnase domain is more stable and is therefore folded and active; the GCN4 domain is substantially unstructured. DNA binding induces folding of GCN4, forcibly unfolding and inactivating the barnase domain. Barnase-GCN4 is thus a "natively unfolded" protein that uses ligand binding to switch between partially folded forms. The key characteristics of each parent protein (catalytic efficiency of barnase, DNA binding affinity and sequence specificity of GCN4) are retained in the chimera. Barnase-GCN4 thus defines a modular approach for assembling enzymes with novel sensor capabilities from a variety of catalytic and ligand binding domains.

Structural characterization of protein unfolding intermediates [Kiefhaber et al. (1995) Nature 375, 513; Hoeltzli et al.(1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9318], which until recently were thought to be nonexistent, is beginning to give information on the mechanism of unfolding. To test for apomyoglobin unfolding intermediates, we monitored kinetics of urea-induced denaturation by stop-flow tryptophan fluorescence and quench-flow amide hydrogen exchange. Both measurements yield a single, measurable kinetic phase of identical rate, indicating that the reaction is highly cooperative. A burst phase in fluorescence, however, suggests that an intermediate is rapidly formed. To structurally characterize it, we carried out stop-flow thiol-disulfide exchange studies of 10 single cysteine-containing mutants. Cysteine probes buried at major sites of helix-helix pairing revealed that side chains throughout the protein unpack and become accessible to the labeling reagent [5, 5'-dithiobis (2-nitrobenzoic acid)] with one of two rates. Probes located at all helical-packing interfaces-except for one-become exposed at the rate of global unfolding as determined by fluorescence and hydrogen exchange measurements. In contrast, probes located at the A-E helical interface undergo complete thiol-disulfide exchange within the mixing dead time of 6 ms. These results point to the existence of a burst-phase unfolding intermediate that contains globally intact hydrogen bonds but locally disrupted side-chain packing interactions. Dissolution of secondary and tertiary structure are therefore not tightly coupled processes. We suggest that disruption of tertiary structure may be a stepwise process that begins at the weakest point of the native fold, as determined by native-state hydrogen-exchange parameters.

It is clear that close-packed side chain interactions play a dominant role in stabilizing native proteins, but the extent to which they stabilize kinetic intermediates and shape the energetic landscape of folding is not known. A method for characterizing structural changes at the level of individual side chains is presented and applied to study the refolding of apomyoglobin mutants containing engineered cysteine residues at key helical packing interfaces. The formation of buried side chain structure at the probe sites is followed by the extent of thiol-disulfide exchange during a pulse of thiol labeling reagent (either methyl methanethiosulfonate or 5,5'-dithiobis (2-nitrobenzoic acid)) applied at various stages of folding. The results suggest that the eight helices pack in at least three distinct stages, involving formation of two intermediates with time constants of

The kinetics of nucleotide-induced changes of tryptophan fluorescence have been measured for recombinant bovine 70 kDa heat shock cognate protein (Hsc70), a 60 kDa subfragment (amino acid residues 1-554) which has ATPase and peptide binding activities, and a 44 kDa subfragment (residues 1-386) which has only ATPase activity. The fluorescence changes resulting from ATP binding to Hsc70 and the 60 kDa fragment are biphasic, and can be interpreted as arising from a two-step process in which ATP initially binds in a bimolecular reaction, followed by a conformational change of the protein-MgATP complex. Fluorescence changes resulting from ADP binding indicate a single-step, bimolecular process. Under single-cycle conditions of the ATPase reaction, a fluorescence change is observed whose rate constant correlates with product release in Hsc70, and with product release/ATP hydrolysis (which are kinetically indistinguishable under single-cycle conditions) in the 60 kDa fragment. These data support a scheme for Hsc70 in which a conformational transition is induced after initial ATP binding but prior to hydrolysis, and the reverse transition is induced by product release. The 60 kDa fragment shows behavior that is quantitatively similar to that of Hsc70. The 44 kDa ATPase fragment does not show biphasic kinetics for ATP binding, and does not show fluorescence changes that suggest conformational changes of the type seen in Hsc70 and the 60 kDa fragment.

Large negative standard heat capacity changes (delta CP degree much less than 0) are the hallmark of processes that remove nonpolar surface from water, including the transfer of nonpolar solutes from water to a nonaqueous phase and the folding, aggregation/association, and ligand-binding reactions of proteins [Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. USA 74, 2236-2240]. More recently, Baldwin [Baldwin, R. L. (1986) Proc. Natl. Acad. Sci. USA 83, 8069-8072] proposed that the delta CP degree of protein folding could be used to quantify the contribution of the burial of nonpolar surface (the hydrophobic effect) to the stability of a globular protein. We demonstrate that identical correlations between the delta CP degree and the change in water-accessible nonpolar surface area (delta Anp) are obtained for both the transfer of nonpolar solutes from water to the pure liquid phase and the folding of small globular proteins: delta CP degree/delta Anp = -(0.28 +/- 0.05) (where delta Anp is expressed in A2 and delta CP degree is expressed in cal.mol-1.K-1; 1 cal = 4.184 J). The fact that these correlations are identical validates the proposals by both Sturtevant and Baldwin that the hydrophobic effect is in general the dominant contributor to delta CP degree and provides a straightforward means of estimating the contribution of the hydrophobic driving force (delta Ghyd degree) to the standard free energy change of a noncovalent process characterized by a large negative delta CP degree in the physiological temperature range: delta Ghyd degree congruent to (80 +/- 10)delta CP degree.

The site-specific binding interaction of lac repressor with a symmetric operator sequence and of EcoRI endonuclease with its specific recognition site both exhibit a characteristic dependence of equilibrium binding constant (Kobs) on temperature, in which Kobs attains a relative maximum in the physiologically relevant temperature range. This behavior, which appears to be quite general for site-specific protein-DNA interactions, is indicative of a large negative standard heat capacity change (delta C0P,obs) in the association process. By analogy with model compound transfer studies and protein folding data, we propose that this delta C0P,obs results primarily from the removal of non-polar surface from water in the association process. From delta C0P,obs we obtain semiquantitative information regarding the change in water-exposed non-polar surface area (delta Anp) and the corresponding hydrophobic driving force for association (delta G0hyd): delta G0hyd approximately equal to 8(+/- 1) x 10(1) delta C0P,obs approximately equal to -22(+/- 5) delta Anp. We propose that removal of non-polar surface from water (the hydrophobic effect) and release of cations (the polyelectrolyte effect) drive the thermodynamically unfavorable process (e.g. conformational distortions) necessary to achieve mutually complementary recognition surfaces (at a steric and functional-group level) in the specific complex.