Measurement of left atrial size as a predictor of severity of illness in sickle cell disease.

Background: Sickle cell disease (SCD) is characterized by microvascular occlusion which leads to multiorgan damage, including left ventricular diastolic dysfunction. Left ventricular diastolic dysfunction has been shown to be an independent risk factor for death in SCD patients. Left atrial dilation (LAD) has been used as a surrogate marker for identification of left ventricular diastolic dysfunction.

Recruitment Strategies in the Integration of Mobile Health Into Sickle Cell Disease Care to Increase Hydroxyurea Utilization Study (meSH): Multicenter Survey Study.

Background: Hydroxyurea is an evidence-based disease-modifying therapy for sickle cell disease (SCD) but is underutilized. The Integration of Mobile Health into Sickle Cell Disease Care to Increase Hydroxyurea Utilization (meSH) multicenter study leveraged mHealth to deliver targeted interventions to patients and providers. SCD studies often underenroll; and recruitment strategies in the SCD population are not widely studied.

Social determinants of health and treatment center affiliation: analysis from the sickle cell disease implementation consortium registry.

Background: Adults with sickle cell disease (SCD) suffer early mortality and high morbidity. Many are not affiliated with SCD centers, defined as no ambulatory visit with a SCD specialist in 2 years. Negative social determinants of health (SDOH) can impair access to care.

"The project did not come to us with a solution": Perspectives of research teams on implementing a study about electronic health record-embedded individualized pain plans for emergency department treatment of vaso-occlusive episodes in adults with sickle cell disease.

Background: This study aimed to capture the implementation process of the ALIGN Study, (An individualized Pain Plan with Patient and Provider Access for Emergency Department care of Sickle Cell Disease). ALIGN aimed to embed Individualized Pain Plans in the electronic health record (E-IPP) and provide access to the plan for both adult patients with sickle cell disease (SCD) and emergency department providers when a person with SCD comes to the emergency department in vaso-occlusive crises.

Methods: Semi-structured interviews were conducted with research teams from the 8 participating sites from the ALIGN study.

Effective Recruitment Strategies for a Sickle Cell Patient Registry Across Sites from the Sickle Cell Disease Implementation Consortium (SCDIC).

Sickle cell disease (SCD) is a genetic disorder predominantly affecting people of African descent and is associated with significant morbidity and mortality. To improve SCD outcomes, the National Heart Lung and Blood Institute funded eight centers to participate in the SCD Implementation Consortium. Sites were required to each recruit 300 individuals with SCD, over 20 months.

Structural, Photophysical, and Photochemical Characterization of Zinc Protoporphyrin IX in a Dimeric Variant of an Iron Storage Protein: Insights into the Mechanism of Photosensitized H Generation.

Some of us have previously reported the preparation of a dimeric form of the iron storage protein, bacterioferritin (Bfr), in which the native heme is substituted with the photosensitizer, Zn(II)-protoporphyrin IX (ZnPP-Bfr dimer). We further showed that the ZnPP-Bfr dimer can serve as a photosensitizer for platinum-catalyzed H generation in aqueous solution without the usually added electron relay between photosensitizer and platinum ( Clark , E. R.

Charge-Disproportionation Symmetry Breaking Creates a Heterodimeric Myoglobin Complex with Enhanced Affinity and Rapid Intracomplex Electron Transfer.

We report rapid photoinitiated intracomplex electron transfer (ET) within a "charge-disproportionated" myoglobin (Mb) dimer with greatly enhanced affinity. Two mutually supportive Brownian Dynamics (BD) interface redesign strategies, one a new "heme-filtering" approach, were employed to "break the symmetry" of a Mb homodimer by pairing Mb constructs with complementary highly positive and highly negative net surface charges, introduced through D/E → K and K → E mutations, respectively. BD simulations using a previously developed positive mutant, Mb(+6) = Mb(D44K/D60K/E85K), led to construction of the complementary negative mutant Mb(-6) = Mb(K45E, K63E, K95E).

Distance-independent charge recombination kinetics in cytochrome c-cytochrome c peroxidase complexes: compensating changes in the electronic coupling and reorganization energies.

Charge recombination rate constants vary no more than 3-fold for interprotein ET in the Zn-substituted wild type (WT) cytochrome c peroxidase (CcP):cytochrome c (Cc) complex and in complexes with four mutants of the Cc protein (i.e., F82S, F82W, F82Y, and F82I), despite large differences in the ET distance.

Evolving the [myoglobin, cytochrome b(5)] complex from dynamic toward simple docking: charging the electron transfer reactive patch.

We describe photoinitiated electron transfer (ET) from a suite of Zn-substituted myoglobin (Mb) variants to cytochrome b(5) (b(5)). An electrostatic interface redesign strategy has led to the introduction of positive charges into the vicinity of the heme edge through D/E → K charge-reversal mutation combinations at "hot spot" residues (D44, D60, and E85), augmented by the elimination of negative charges from Mb or b(5) by neutralization of heme propionates. These variations create an unprecedentedly large range in the product of the ET partners' total charges (-5 < -q(Mb)q(b(5)) < 40).

Interfacial hydration, dynamics and electron transfer: multi-scale ET modeling of the transient [myoglobin, cytochrome b5] complex.

Formation of a transient [myoglobin (Mb), cytochrome b(5) (cyt b(5))] complex is required for the reductive repair of inactive ferri-Mb to its functional ferro-Mb state. The [Mb, cyt b(5)] complex exhibits dynamic docking (DD), with its cyt b(5) partner in rapid exchange at multiple sites on the Mb surface. A triple mutant (Mb(3M)) was designed as part of efforts to shift the electron-transfer process to the simple docking (SD) regime, in which reactive binding occurs at a restricted, reactive region on the Mb surface that dominates the docked ensemble.

Faster interprotein electron transfer in a [myoglobin, b⁵] complex with a redesigned interface.

Direct measurements of electron transfer (ET) within a protein-protein complex with a redesigned interface formed by physiological partner proteins myoglobin (Mb) and cytochrome b(5) (b(5)) reveal interprotein ET rates comparable to those observed within the photosynthetic reaction center. Brownian dynamics simulations show that Mb in which three surface acid residues are mutated to lysine binds b(5) in an ensemble of configurations distributed around a reactive most-probable structure. Correspondingly, charge-separation ET from a photoexcited singlet zinc porphyrin incorporated within Mb to the heme of b(5) and the follow-up charge-recombination exhibit distributed kinetics, with median rate constants, k(f)(s) = 2.

Photoinitiated singlet and triplet electron transfer across a redesigned [myoglobin, cytochrome b5] interface.

We describe a strategy by which reactive binding of a weakly bound, 'dynamically docked (DD)' complex without a known structure can be strengthened electrostatically through optimized placement of surface charges, and discuss its use in modulating complex formation between myoglobin (Mb) and cytochrome b(5) (b(5)). The strategy employs paired Brownian dynamics (BD) simulations, one which monitors overall binding, the other reactive binding, to examine [X --> K] mutations on the surface of the partners, with a focus on single and multiple [D/E --> K] charge reversal mutations. This procedure has been applied to the [Mb, b(5)] complex, indicating mutations of Mb residues D44, D60, and E85 to be the most promising, with combinations of these showing a nonlinear enhancement of reactive binding.

Electrostatic redesign of the [myoglobin, cytochrome b5] interface to create a well-defined docked complex with rapid interprotein electron transfer.

Cyt b(5) is the electron-carrier "repair" protein that reduces met-Mb and met-Hb to their O(2)-carrying ferroheme forms. Studies of electron transfer (ET) between Mb and cyt b(5) revealed that they react on a "Dynamic Docking" (DD) energy landscape on which binding and reactivity are uncoupled: binding is weak and involves an ensemble of nearly isoenergetic configurations, only a few of which are reactive; those few contribute negligibly to binding. We set the task of redesigning the surface of Mb so that its reaction with cyt b(5) instead would occur on a conventional "simple docking" (SD) energy landscape, on which a complex exhibits a well-defined (set of) reactive binding configuration(s), with binding and reactivity thus no longer being decoupled.

Kinetic-dynamic model for conformational control of an electron transfer photocycle: mixed-metal hemoglobin hybrids.

It is becoming increasingly clear that the transfer of an electron across a protein-protein interface is coupled to the dynamics of conformational conversion between and within ensembles of interface conformations. Electron transfer (ET) reactions in conformationally mobile systems provide a "clock" against which the rapidity of a dynamic process may be measured, and we here report a simple kinetic (master equation) model that self-consistently incorporates conformational dynamics into an ET photocycle comprised of a photoinitiated "forward" step and thermal return to ground. This kinetic/dynamic (KD) model assumes an ET complex exists as multiple interconverting conformations which partition into an ET-optimized (reactive; R) population and a less-reactive population ( S).

Dynamic docking of cytochrome b5 with myoglobin and alpha-hemoglobin: heme-neutralization "squares" and the binding of electron-transfer-reactive configurations.

Intracomplex electron transfer (ET) occurs most often in intrinsically transient, low affinity complexes. As a result, the means by which adequate specificity and reactivity are obtained to support effective ET is still poorly understood. We report here on two such ET complexes: cytochrome b5 (cyt b5) in reaction with its physiological partners, myoglobin (Mb) and hemoglobin (Hb).

NMR spectroscopy can characterize proteins encapsulated in a sol-gel matrix.

Proteins encapsulated within sol-gel matrices (SG) have the potential to fill many scientific and technological roles, but these applications are hindered by the limited means of probing possible structural consequences of encapsulation. We here present the first demonstration that it is possible to obtain high-resolution, solution NMR measurements of proteins encapsulated within a SG matrix. With the aim of determining the breadth of this approach, we have encapsulated three paramagnetic proteins with different overall charges: the highly acidic human Fe3+ cytochrome b5 (cyt b5); the highly basic horse heart cytochrome c (cyt c); and the nearly neutral, sperm whale cyanomet-myoglobin.

Equilibrium/nonequilibrium initial configurations in forward/reverse electron transfer within mixed-metal hemoglobin hybrids.

In a protein-protein electron transfer (ET) photocycle, the "forward" ET reaction is initiated with the excited complex, [3DA], in an equilibrium ensemble of configurations, the majority of which exhibit less than the maximal ET matrix element. In contrast, the charge-separated intermediate complex is formed in a nonequilibrium set of configurations with maximal ET matrix elements and would be expected to return to the ground state with the largest rate constant possible unless conformational interconversion first "breaks the connection" and the complex converts to less-reactive substates. According to this analysis, the forward and back ET reactions should show a differential response to viscosity, and the latter could even show an increased rate constant under conditions which suppress departure from the reactive configuration(s).

Hopping in the electron-transfer photocycle of the 1:1 complex of Zn-cytochrome c peroxidase with cytochrome c.

The physiological electron-transfer (ET) partners, cytochrome c peroxidase (CcP) and cytochrome c (Cc)1, can be modified to exhibit photoinitiated ET through substitution of Zn (or Mg) for Fe in either partner. Laser excitation of the Zn-porphyrin (ZnP) to its triplet excited state (3ZnP) initiates direct heme-heme ET to the ferriheme center of its partner across the protein-protein interface. This photoinitiated ET produces the charge-separated intermediate, I = [ZnP+CcP, Fe2+Cc], with a metalloporphyrin pi-cation radical (ZnP+) in the donor protein and a ferroheme acceptor protein.

Differential influence of dynamic processes on forward and reverse electron transfer across a protein-protein interface.

We propose that the forward and reverse halves of a flash-induced protein-protein electron transfer (ET) photocycle should exhibit differential responses to dynamic interconversion of configurations when the most stable configuration is not the most reactive, because the reactants exist in different initial configurations: the flash-photoinitiated forward ET process begins with the protein partners in an equilibrium ensemble of configurations, many of which have little or no reactivity, whereas the reactant of the thermal back ET (the charge-separated intermediate) is formed in a nonequilibrium, "activated" protein configuration. We report evidence for this proposal in measurements on (i) mixed-metal hemoglobin hybrids, (ii) the complex between cytochrome c peroxidase and cytochrome c, and (iii and iv) the complexes of myoglobin and isolated hemoglobin alpha-chains with cytochrome b(5). For all three systems, forward and reverse ET does respond differently to modulation of dynamic processes; further, the response to changes in viscosity is different for each system.

Electrostatic influence on rotational mobilities of sol-gel-encapsulated solutes by NMR and EPR spectroscopies.

The rotational mobilities of small solute molecules encapsulated in tetramethyl orthosilicate (TMOS) sol-gels have been investigated by EPR spectroscopy of encapsulated nitroxide probes and by high-resolution NMR spectroscopic measurements of transferred NOE's (trNOE's), of T(1)'s, and of T(1)'s in the rotating frame (T(1)rho). The two spectroscopic methods are sensitive to motions on different time scales and hence, are nicely complementary. Suites of neutral, positively, and negatively charged nitroxide probes (EPR) and of simple diamagnetic small molecules (NMR) were selected to disclose influences of electrostatic interactions with the sol-gel walls and to probe the presence of multiple populations of molecules in distinct regions of the sol-gel pores.

Conformational substates of the oxyheme centers in alpha and beta subunits of hemoglobin as disclosed by EPR and ENDOR studies of cryoreduced protein.

Exposure of frozen solutions of oxyhemoglobin to gamma-irradiation at 77 K yields EPR- and ENDOR-active, one-electron-reduced oxyheme centers which retain the conformation of the diamagnetic precursor. EPR spectra have been collected for the centers produced in human HbO(2) and isolated alphaO(2) and betaO(2) chains, as well as alphaO(2)beta(Zn), alpha(Zn)betaO(2), and alphaO(2)beta(Fe(3+)) hybrids, each in frozen buffer and in frozen glasses that form in the presence of glycols and sugars and also in the presence of IHP. These reveal two spectroscopically distinct classes of such ferriheme centers (g(1) View Article and Find Full Text PDF

Dynamic docking and electron-transfer between cytochrome b5 and a suite of myoglobin surface-charge mutants. Introduction of a functional-docking algorithm for protein-protein complexes.

Horse myoglobin (Mb) provides a convenient "workbench" for probing the effects of electrostatics on binding and reactivity in the dynamic [Mb, cytochrome b(5)] electron-transfer (ET) complex. We have combined mutagenesis and heme neutralization to prepare a suite of six Mb surface-charge variants: the [S92D]Mb and [V67R]Mb mutants introduce additional charges on the "front" face, and incorporation of the heme di-ester into each of these neutralizes the charge on the heme propionates which further increases the positive charge on the "front" face. For this set of mutants, the nominal charge of Mb changes by -1 to +3 units relative to that for native Mb.

Myoglobin and cytochrome b5: a nuclear magnetic resonance study of a highly dynamic protein complex.

The transient complex of bovine myoglobin and cytochrome b(5) has been investigated using a combination of NMR chemical shift mapping, (15)N relaxation data, and protein docking simulations. Chemical shift perturbations observed for cytochrome b(5) amide resonances upon complex formation with either metmyoglobin (Fe(III)) or carbon monoxide-bound myoglobin (Fe(II)) are more than 10-fold smaller than in other transient redox protein complexes. From (15)N relaxation experiments, an increase in the overall correlation time of cytochrome b(5) in the presence of myoglobin is observed, confirming that complex formation is occurring.

Interprotein electron transfer in a confined space: uncoupling protein dynamics from electron transfer by sol-gel encapsulation.

In this paper, we describe the first observations of photoinitiated interprotein electron transfer (ET) within sol-gels. We have encapsulated three protein-protein complexes, specifically selected because they represent a full range of affinities, are sensitive to different types of dynamic processes, and thus are expected to respond differently to sol-gel encapsulation. The three systems are (i) the [Zn, Fe(3+)L] mixed-metal hemoglobin hybrids, where the alpha(1)-Zn and beta(2)-Fe subunits correspond to a "predocked" protein-protein complex with a crystallographically defined interface (Natan, M.

Dynamic docking and electron transfer between Zn-myoglobin and cytochrome b(5).

We present a broad study of the effect of neutralizing the two negative charges of the Mb propionates on the interaction and electron transfer (ET) between horse Mb and bovine cyt b(5), through use of Zn-substituted Mb (ZnMb, 1) to study the photoinitiated reaction, ((3)ZnP)Mb + Fe(3+)cyt b(5) --> (ZnP)(+)Mb + Fe(2+)cyt b(5). The charge neutralization has been carried out both by replacing the Mb heme with zinc-deuteroporphyrin dimethylester (ZnMb(dme), 2), which replaces the charges by small neutral hydrophobic patches, and also by replacement with the newly prepared zinc-deuteroporphyrin diamide (ZnMb(diamide), 3), which converts the charged groups to neutral, hydrophilic ones. The effect of propionate neutralization on the conformation of the zinc-porphyrin in the Mb heme pocket has been studied by multinuclear NMR with an (15)N labeled zinc porphyrin derivative (ZnMb((15)N-diamide), 4).