Point-of-care sensors, which are low-cost and user-friendly, play a crucial role in precision medicine by providing quick results for individuals. Here, we transform the conventional glucometer into a 4-hydroxytamoxifen therapeutic biosensor in which 4-hydroxytamoxifen modulates the electrical signal generated by glucose oxidation. To encode the 4-hydroxytamoxifen signal within glucose oxidation, we introduce the ligand-binding domain of estrogen receptor-alpha into pyrroloquinoline quinone-dependent glucose dehydrogenase by constructing and screening a comprehensive protein insertion library. In addition to obtaining 4-hydroxytamoxifen regulatable engineered proteins, these results unveil the significance of both secondary and quaternary protein structures in propagation of conformational signals. By constructing an effective bioelectrochemical interface, we detect 4-hydroxytamoxifen in human blood samples as changes in the electrical signal and use this to develop an electrochemical algorithm to decode the 4-hydroxytamoxifen signal from glucose. To meet the miniaturization and signal amplification requirements for point-of-care use, we harness power from glucose oxidation to create a self-powered sensor. We also amplify the 4-hydroxytamoxifen signal using an organic electrochemical transistor, resulting in milliampere-level signals. Our work demonstrates a broad interdisciplinary approach to create a biosensor that capitalizes on recent innovations in protein engineering, electrochemical sensing, and electrical engineering.

Abstract Precision medicine is expected to revolutionize healthcare by prioritizing accuracy and efficacy over the traditional "one-fits-all" approach. Point-of-care (POC) sensors, which are low-cost and user-friendly, play a crucial role in driving this trend by providing quick results for individuals. Modeled after the 5G network, we conceptualized an innovative approach for transmitting biomolecular signals - encoding biomolecular binding by modulating the electrical signal from glucose oxidation. We implement this concept by engineering a hybrid protein that incorporates a biomarker binding domain within a glucose oxidoreductase. By constructing an effective bioelectrochemical interface, we could detect 4-hydroxytamoxifen, an estrogen antagonist, in human blood samples, as real-time changes in the electrical signal. Moreover, our design uses blood glucose to power this real-time sensor and an additional transistor, which yields a self-powered prototype with high signal-to-noise. We foresee this novel approach transforming the conventional glucometer into a therapeutic biosensor with add-in functions.

Abstract Precision medicine is expected to revolutionize healthcare by prioritizing accuracy and efficacy over the traditional "one-fits-all" approach. Point-of-care (POC) sensors, which are low-cost and user-friendly, play a crucial role in driving this trend by providing quick results for individuals. Modeled after the 5G network, we conceptualized an innovative approach for transmitting biomolecular signals - encoding biomolecular binding by modulating the electrical signal from glucose oxidation. We implement this concept by engineering a hybrid protein that incorporates a biomarker binding domain within a glucose oxidoreductase. By constructing an effective bioelectrochemical interface, we could detect 4-hydroxytamoxifen, an estrogen antagonist, in human blood samples, as real-time changes in the electrical signal. Moreover, our design uses blood glucose to power this real-time sensor and an additional transistor, which yields a self-powered prototype with high signal-to-noise. We foresee this novel approach transforming the conventional glucometer into a therapeutic biosensor with add-in functions.

Engineered living materials (ELMs) embed living cells in a biopolymer matrix to create materials with tailored functions. While bottom-up assembly of macroscopic ELMs with a de novo matrix would offer the greatest control over material properties, we lack the ability to genetically encode a protein matrix that leads to collective self-organization. Here we report growth of ELMs from Caulobacter crescentus cells that display and secrete a self-interacting protein. This protein formed a de novo matrix and assembled cells into centimeter-scale ELMs. Discovery of design and assembly principles allowed us to tune the composition, mechanical properties, and catalytic function of these ELMs. This work provides genetic tools, design and assembly rules, and a platform for growing ELMs with control over both matrix and cellular structure and function.

After reacting with hydrogen peroxide (H2O2), sickle-cell hemoglobin (HbS, βE6V) remains longer in a highly oxidizing ferryl form (HbFe4+=O) and induces irreversible oxidation of “hot-spot” amino acids, including βCys-93. To control the damaging ferryl heme, here we constructed three HbS variants. The first contained a redox-active Tyr in β subunits (F41Y), a substitution present in Hb Mequon; the second contained the Asp (K82D) found in the β cleft of Hb Providence; and the third had both of these β substitutions. Both the single Tyr-41 and Asp-82 constructs lowered the oxygen affinity of HbS but had little or no effects on autoxidation or heme loss kinetics. In the presence of H2O2, both rHbS βF41Y and βF41Y/K82D enhanced ferryl Hb reduction by providing a pathway for electrons to reduce the heme via the Tyr-41 side chain. MS analysis of βCys-93 revealed moderate inhibition of thiol oxidation in the HbS single F41Y variant and dramatic 3- to 8-fold inhibition of cysteic acid formation in rHbS βK82D and βF41Y/K82D, respectively. Under hypoxia, βK82D and βF41Y/K82D HbS substitutions increased the delay time by ∼250 and 600 s before the onset of polymerization compared with the rHbS control and rHbS βF41Y, respectively. Moreover, at 60 °C, rHbS βK82D exhibited superior structural stability. Asp-82 also enhanced the function of Tyr as a redox-active amino acid in the rHbS βF41Y/K82D variant. We conclude that the βK82D and βF41Y substitutions add significant resistance to oxidative stress and anti-sickling properties to HbS and therefore could be potential genome-editing targets.

Tautomycetin (TTN) is a polyketide natural product featuring a terminal alkene. Functional characterization of the genes within the ttn gene cluster from Streptomyces griseochromogenes established the biosynthesis of the TTN polyketide backbone, its dialkylmaleic anhydride moiety, the coupling of the two moieties to form the nascent intermediate TTN F-1, and the tailoring steps converting TTN F-1 to TTN. Here, we report biochemical and structural characterization of TtnD, a prenylated FMN (prFMN)-dependent decarboxylase belonging to the UbiD family that catalyzes the penultimate step of TTN biosynthesis. TtnD catalyzes decarboxylation of TTN D-1 to TTN I-1, utilizing prFMN as a cofactor generated by the TtnC flavin prenyltransferase; both TtnD and TtnC are encoded within the ttn biosynthetic gene cluster. TtnD exhibits substrate promiscuity but accepts only TTN D-1 congeners that feature an α,β-unsaturated acid, supporting the [3+2] cycloaddition mechanism during catalysis that requires the double bond of an α,β-unsaturated acid substrate. TtnD shares a similar overall structure with other members of the UbiD family but forms a homotetramer in solution. Each protomer is composed of three domains with the active site located between the middle and C-terminal domains; R169-E272-E277, constituting the catalytic triad, and E228, involved in Mn(II)-mediated binding of prFMN, were confirmed by site-directed mutagenesis. TtnD represents the first example of a prFMN-dependent decarboxylase involved in polyketide biosynthesis, expanding the substrate scope of the UbiD family of decarboxylases beyond simple aromatic and cinnamic acids. TtnD and its homologues are widespread in nature and could be exploited as biocatalysts for organic synthesis.

Mutations in hemoglobin can cause a wide range of phenotypic outcomes, including anemia due to protein instability and red cell lysis. Uncovering the biochemical basis for these phenotypes can provide new insights into hemoglobin structure and function as well as identify new therapeutic opportunities. We report here a new hemoglobin α chain variant in a female patient with mild anemia, whose father also carries the trait and is from the Turkish city of Kirklareli. Both the patient and her father had a His-58(E7) → Leu mutation in α1. Surprisingly, the patient's father is not anemic, but he is a smoker with high levels of HbCO (∼16%). To understand these phenotypes, we examined recombinant human Hb (rHb) Kirklareli containing the α H58L replacement. Mutant α subunits containing Leu-58(E7) autoxidize ∼8 times and lose hemin ∼200 times more rapidly than native α subunits, causing the oxygenated form of rHb Kirklareli to denature very rapidly under physiological conditions. The crystal structure of rHb Kirklareli shows that the α H58L replacement creates a completely apolar active site, which prevents electrostatic stabilization of bound O2, promotes autoxidation, and enhances hemin dissociation by inhibiting water coordination to the Fe(III) atom. At the same time, the mutant α subunit has an ∼80,000-fold higher affinity for CO than O2, causing it to rapidly take up and retain carbon monoxide, which prevents denaturation both in vitro and in vivo and explains the phenotypic differences between the father, who is a smoker, and his daughter.

Previous work suggested that hemoglobin (Hb) tetramer formation slows autoxidation and hemin loss and that the naturally occurring mutant, Hb Providence (HbProv; βK82D), is much more resistant to degradation by H2O2 We have examined systematically the effects of genetic cross-linking of Hb tetramers with and without the HbProv mutation on autoxidation, hemin loss, and reactions with H2O2, using native HbA and various wild-type recombinant Hbs as controls. Genetically cross-linked Hb Presbyterian (βN108K) was also examined as an example of a low oxygen affinity tetramer. Our conclusions are: (a) at low concentrations, all the cross-linked tetramers show smaller rates of autoxidation and hemin loss than HbA, which can dissociate into much less stable dimers and (b) the HbProv βK82D mutation confers more resistance to degradation by H2O2, by markedly inhibiting oxidation of the β93 cysteine side chain, particularly in cross-linked tetramers and even in the presence of the destabilizing Hb Presbyterian mutation. These results show that cross-linking and the βK82D mutation do enhance the resistance of Hb to oxidative degradation, a critical element in the design of a safe and effective oxygen therapeutic.

Water molecules can enter the heme pockets of unliganded myoglobins and hemoglobins, hydrogen bond with the distal histidine, and introduce steric barriers to ligand binding. The spectrokinetics of photodissociated CO complexes of human hemoglobin and its isolated α and β chains were analyzed for the effect of heme hydration on ligand rebinding. A strong coupling was observed between heme hydration and quaternary state. This coupling may contribute significantly to the 20-60-fold difference between the R- and T-state bimolecular CO binding rate constants and thus to the modulation of ligand reactivity that is the hallmark of hemoglobin allostery. Heme hydration proceeded over the course of several kinetic phases in the tetramer, including the R to T quaternary transition. An initial 150 ns hydration phase increased the R-state distal pocket water occupancy, nw(R), to a level similar to that of the isolated α (∼60%) and β (∼10%) chains, resulting in a modest barrier to ligand binding. A subsequent phase, concurrent with the first step of the R → T transition, further increased the level of heme hydration, increasing the barrier. The final phase, concurrent with the final step of the allosteric transition, brought the water occupancy of the T-state tetramer, nw(T), even higher and close to full occupancy in both the α and β subunits (∼90%). This hydration level could present an even larger barrier to ligand binding and contribute significantly to the lower iron reactivity of the T state toward CO.

A pathogenic V67M mutation occurs at the E11 helical position within the heme pockets of variant human fetal and adult hemoglobins (Hb). Subsequent post-translational modification of Met to Asp was reported in gamma subunits of human fetal Hb Toms River (gamma 67(E11) Val -> Met) and beta subunits of adult Hb (HbA) Bristol-Alesha (beta 67(E11) Val -> Met) that were associated with hemolytic anemia. Using kinetic, proteomic, and crystal structural analysis, we were able to show that the Met -> Asp transformation involves heme cycling through its oxoferryl state in the recombinant versions of both proteins. The conversion to Met and Asp enhanced the spontaneous autoxidation of the mutants relative to wild-type HbA and human fetal Hb, and the levels of Asp were elevated with increasing levels of hydrogen peroxide (H2O2). Using H-2 O-18(2), we verified incorporation of (18)Ointo the Asp carboxyl side chain confirming the role ofH(2)O(2) in the oxidation of the Met side chain. Under similar experimental conditions, there was no conversion to Asp at the alpha Met(E11) position in the correspondingHbAEvans (alpha 62(E11) Val -> Met). The crystal structures of the three recombinant Met(E11) mutants revealed similar thioether side chain orientations. However, asinthesolutionexperiments, autoxidationof theHbmutantcrystals leads to electron density maps indicative of Asp(E11) formation in beta subunits but not in alpha subunits. This novel post-translational modification highlights the nonequivalence of human Hb alpha, beta, and gamma subunits with respect to redox reactivity and may have direct implications to alpha/beta hemoglobinopathies and design of oxidatively stable Hb-based oxygen therapeutics.

We have developed the method of picosecond Laue crystallography and used this capability to probe ligand dynamics in tetrameric R-state hemoglobin (Hb). Time-resolved, 2 Å-resolution electron density maps of photolyzed HbCO reveal the time-dependent population of CO in the binding (A) and primary docking (B) sites of both α and β subunits from 100 ps to 10 μs. The proximity of the B site in the β subunit is about 0.25 Å closer to its A binding site, and its kBA rebinding rate (~300 μs-1) is six times faster, suggesting distal control of the rebinding dynamics. Geminate rebinding in the β subunit exhibits both prompt and delayed geminate phases. We developed a microscopic model to quantitatively explain the observed kinetics, with three states for the α subunit and four states for the β subunit. This model provides a consistent framework for interpreting rebinding kinetics reported in prior studies of both HbCO and HbO2.

Although molecular dynamics simulations suggest multiple interior pathways for O2 entry into and exit from globins, most experiments indicate well defined single pathways. In 2001, we highlighted the effects of large-to-small amino acid replacements on rates for ligand entry and exit onto the three-dimensional structure of sperm whale myoglobin. The resultant map argued strongly for ligand movement through a short channel from the heme iron to solvent that is gated by the distal histidine (His-64(E7)) near the solvent edge of the porphyrin ring. In this work, we have applied the same mutagenesis mapping strategy to the neuronal mini-hemoglobin from Cerebratulus lacteus (CerHb), which has a large internal tunnel from the heme iron to the C-terminal ends of the E and H helices, a direction that is 180° opposite to the E7 channel. Detailed comparisons of the new CerHb map with expanded results for Mb show unambiguously that the dominant (>90%) ligand pathway in CerHb is through the internal tunnel, and the major (>75%) ligand pathway in Mb is through the E7 gate. These results demonstrate that: 1) mutagenesis mapping can identify internal pathways when they exist; 2) molecular dynamics simulations need to be refined to address discrepancies with experimental observations; and 3) alternative pathways have evolved in globins to meet specific physiological demands.

His(E7) to Trp replacements in HbA lead to markedly biphasic bimolecular CO rebinding after laser photolysis. For isolated mutant subunits, the fraction of fast phase increases with increasing [CO], suggesting a competition between binding to an open conformation with an empty E7 channel and relaxation to blocked or closed, slowly reacting states. The rate of conformational relaxation of the open state is ∼18,000 s−1 in α subunits and ∼10-fold faster in β subunits, ∼175,000 s−1. Crystal structures were determined for tetrameric α(WT)β(Trp-63) HbCO, α(Trp-58)β(WT) deoxyHb, and Trp-64 deoxy- and CO-Mb as controls. In Trp-63(E7) βCO, the indole side chain is located in the solvent interface, blocking entry into the E7 channel. Similar blocked Trp-64(E7) conformations are observed in the mutant Mb crystal structures. In Trp-58(E7) deoxy-α subunits, the indole side chain fills both the channel and the distal pocket, forming a completely closed state. The bimolecular rate constant for CO binding, k′CO, to the open conformations of both mutant Hb subunits is ∼80–90 μm−1 s−1, whereas k′CO for the completely closed states is 1000-fold slower, ∼0.08 μm−1 s−1. A transient intermediate with k′CO ≈ 0.7 μm−1 s−1 is observed after photolysis of Trp-63(E7) βCO subunits and indicates that the indole ring blocks the entrance to the E7 channel, as observed in the crystal structures of Trp(E7) deoxyMb and βCO subunits. Thus, either blocking or completely filling the E7 channel dramatically slows bimolecular binding, providing strong evidence that the E7 channel is the major pathway (≥90%) for ligand entry in human hemoglobin.

To generate a deeper understanding into the relations between protein structure, dynamics, and function, we have developed time-resolved X-ray methods capable of probing changes in protein structure on time scales as short as ∼100 ps. This infrastructure was first developed on the ID09B time-resolved beamline at the ESRF, and more recently at the ID14B BioCARS beamline at the APS. In these studies, a picosecond laser pulse first photoexcites a protein and triggers a structural change, then a suitably delayed picosecond X-ray pulse passes through the protein and the scattered X-rays are imaged on a 2D detector. When the sample is a protein crystal, this “pump-probe” approach recovers time-resolved diffraction “snapshots” whose corresponding electron density maps can be stitched together into movies that unveil the correlated motions that arise from the photoexcitation event. When the sample is a protein solution, we recover time-resolved small- and wide-angle X-ray scattering (SAXS/WAXS) patterns that are sensitive to changes in the size, shape, and structure of the protein. The time-resolved x-ray scattering diffractometer developed at the APS is capable of simultaneously probing both SAXS and WAXS regions with 100 ps time resolution. Importantly, scattering studies of proteins in solution unveil structural dynamics without the constraints imposed by crystal contacts; thus, these scattering “fingerprints” complement results obtained from diffraction studies. Together, these studies provide stringent constraints for putative models of conformational states and structural transitions between them. Time-resolved studies of heme proteins have unveiled at an unprecedented level of structural detail protein conformational dynamics as well as ligand migration and escape. This research was supported in part by the Intramural Research Program of the NIH, NIDDK. JSO is supported by NIH grants GM035469 and HL047020 and Grant C0612 from the Robert Welch A. Foundation.

Water molecules in internal protein cavities play fundamental roles in satisfying the H-bonding potentials of main chain atoms in turns, coils, and loops, determining the stability and rigidity of proteins, shifting the pKa values of buried ionizable residues, and modulating dynamical processes such as folding, catalysis, and proton transfers. Detecting these internal water molecules is sometimes obscured in x-ray crystallography due to positional disorder. We have developed a spectrokinetic assay that accurately detects the presence of a non-coordinated water molecule in the distal heme pocket of myoglobin and in a series of distal pocket mutants, including many where this water molecule is positionally disordered. We also have shown that this water plays a major role in determining the observed bimolecular recombination rate constant. We show that 1) this water molecule modulates the ligand binding dynamics of a series of H64, L29 and V68 mutants; 2) it plays the major role in the observed pH dependence of the CO recombination kinetics between pH 4 and 7 with the protonation of the distal histidine acting as a switch to change water occupancy; and 3) it may also modulate ligand binding dynamics in isolated hemoglobin chains, with the occupancy being larger in the alpha chains. Accurately measuring water occupancy in heme proteins answers crucial questions about water in apolar or slightly polar protein cavities and clarifies the role internal water molecules play in modulating protein function.

The entry of a water molecule into the distal heme pocket of pentacoordinate heme proteins such as myoglobin and the alpha,beta chains of hemoglobin can be detected by time-resolved spectroscopy in the heme visible bands after photolysis of the CO complex. Reviewing the evidence from spectrokinetic studies of Mb variants, we find that this optical method measures the occupancy of non(heme)coordinated water in the distal pocket, n(w), with high fidelity. This evidence further suggests that perturbation of the kinetic barrier presented by distal pocket water is often the dominant mechanism by which active site mutations affect the bimolecular rate constant for CO binding. Water entry into the heme pockets of isolated hemoglobin subunits was detected by optical methods. Internal hydration is higher in the native alpha chains than in the beta chains, in agreement with previous crystallographic results for the subunits within Hb tetramers. The kinetic parameters obtained from modeling of the water entry and ligand rebinding in Mb mutants and native Hb chains are consistent with an inverse dependence of the bimolecular association rate constant on the water occupancy factor. This correlation suggests that water and ligand mutually exclude one another from the distal pockets of both types of hemoglobin chains and myoglobin.

Internal water molecules are important to protein structure and function, but positional disorder and low occupancies can obscure their detection by X-ray crystallography. Here, we show that water can be detected within the distal cavities of myoglobin mutants by subtle changes in the absorbance spectrum of pentacoordinate heme, even when the presence of solvent is not readily observed in the corresponding crystal structures. A well-defined, noncoordinated water molecule hydrogen bonded to the distal histidine (His64) is seen within the distal heme pocket in the crystal structure of wild type (wt) deoxymyoglobin. Displacement of this water decreases the rate of ligand entry into wt Mb, and we have shown previously that the entry of this water is readily detected optically after laser photolysis of MbCO complexes. However, for L29F and V68L Mb no discrete positions for solvent molecules are seen in the electron density maps of the crystal structures even though His64 is still present and slow rates of ligand binding indicative of internal water are observed. In contrast, time-resolved perturbations of the visible absorption bands of L29F and V68L deoxyMb generated after laser photolysis detect the entry and significant occupancy of water within the distal pockets of these variants. Thus, the spectral perturbation of pentacoordinate heme offers a potentially robust system for measuring nonspecific hydration of the active sites of heme proteins.

A detailed mechanistic understanding of how a protein functions requires knowledge not only of its static structure, but also how its conformation evolves as it executes its function. The recent development of picosecond time-resolved X-ray crystallography has allowed us to visualize in real time and with atomic detail the conformational evolution of a protein. Here, we report the photolysis-induced structural evolution of wild-type and L29F myoglobin over times ranging from 100 ps to 3 μs. The sub-ns structural rearrangements that accompany ligand dissociation in wild-type and the mutant form differ dramatically, and lead to vastly different ligand migration dynamics. The correlated protein displacements provide a structural explanation for the kinetic differences. Our observation of functionally important protein motion on the sub-ns time scale was made possible by the 150-ps time resolution of the measurement, and demonstrates that picosecond dynamics are relevant to protein function. To visualize subtle structural changes without modeling, we developed a novel method for rendering time-resolved electron density that depicts motion as a color gradient across the atom or group of atoms that move. A sequence of these time-resolved images have been stitched together into a movie, which allows one to literally “watch” the protein as it executes its function.