Phytochromes are biological red/far-red light sensors found in many organisms. Photoisomerization of the linear methine-bridged tetrapyrrole triggers transient proton translocation events in the chromophore binding pocket (CBP) leading to major conformational changes of the protein matrix that are in turn associated with signaling. By combining pH-dependent resonance Raman and UV-visible absorption spectroscopy, we analyzed protonation-dependent equilibria in the CBP of Cph1 involving the proposed Pr-I and Pr-II substates that prevail below and above pH 7.5, respectively. The protonation pattern and vibrational properties of these states were further characterized by means of hybrid quantum mechanics/molecular mechanics calculations. From this combined experimental-theoretical study, we were able to identify His260 as the key residue controlling pH-dependent equilibria. This residue is not only responsible for the conformational heterogeneity of CBP in the Pr state of prokaryotic phytochromes, discussed extensively in the past, but it constitutes the sink and source of protons in the proton release/uptake mechanism involving the tetrapyrrole chromophore which finally leads to the formation of the Pfr state. Thus, this work provides valuable information that may guide further experiments toward the understanding of the specific role of protons in controlling structure and function of phytochromes in general.
Widely used diagnostic tools make use of antibodies recognizing targeted molecules, but additional techniques are required in order to alleviate the disadvantages of antibodies. Herein, molecular dynamic calculations are performed for the design of high affinity artificial protein binding surfaces for the recognition of neuron specific enolase (NSE), a known cancer biomarker. Computational simulations are employed to identify particularly stabile secondary structure elements. These epitopes are used for the subsequent molecular imprinting, where surface imprinting approach is applied. The molecular imprints generated with the calculated epitopes of greater stability (Cys‐Ep1) show better binding properties than those of lower stability (Cys‐Ep5). The average binding strength of imprints created with stabile epitopes is found to be around twofold and fourfold higher for the NSE derived peptide and NSE protein, respectively. The recognition of NSE is investigated in a wide concentration range, where high sensitivity (limit of detection (LOD) = 0.5 ng mL−1) and affinity (dissociation constant (Kd) = 5.3 × 10−11m) are achieved using Cys‐Ep1 imprints reflecting the stable structure of the template molecules. This integrated approach employing stability calculations for the identification of stabile epitopes is expected to have a major impact on the future development of high affinity protein capturing binders.
Similar reactions of 2,6-dipicolinoylbis(N,N-diethylthiourea) (H2L(a)) with: (i) Ni(NO3)2·6H2O, (ii) a mixture of Ni(NO3)2·6H2O and AgNO3, (iii) a mixture of Ni(OAc)2·4H2O and PrCl3·7H2O and (iv) a mixture of Ni(OAc)2·4H2O and BaCl2·2H2O give the binuclear complex [Ni2(L(a))2(MeOH)(H2O)], the polymeric compound [NiAg2(L(a))2]∞, and the heterobimetallic complexes [Ni2Pr(L(a))2(OAc)3] and [Ni2Ba(L(a))3], respectively. The obtained assemblies can be used for the build up of supramolecular polymers by means of weak and medium intermolecular interactions. Two prototype examples of such compounds, which are derived from the trinuclear complexes of the types [MLn(III)(L)2(OAc)3] and [MBa(L)3], are described with the compounds {[CuDy(III)(L(a))2(p-O2C-C6H4-CO2)(MeOH)4]Cl}∞ and [MnBa(MeOH)(L(b))3]∞, H2L(b) = 2,6-dipicolinoylbis(N,N-morpholinoylthiourea).
Agp1 is a prototypical bacterial phytochrome from Agrobacterium fabrum harboring a biliverdin cofactor which reversibly photoconverts between a red-light-absorbing (Pr) and a far-red-light-absorbing (Pfr) states. The reaction mechanism involves the isomerization of the bilin-chromophore followed by large structural changes of the protein matrix that are coupled to protonation dynamics at the chromophore binding site. Histidines His250 and His280 participate in this process. Although the three-dimensional structure of Agp1 has been solved at high resolution, the precise position of hydrogen atoms and protonation pattern in the chromophore binding pocket has not been investigated yet. Here, we present protonated structure models of Agp1 in the Pr state involving appropriately placed hydrogen atoms that were generated by hybrid quantum mechanics/molecular mechanics- and electrostatic calculations and validated against experimental structural- and spectroscopic data. Although the effect of histidine protonation on the vibrational spectra is weak, our results favor charge neutral H250 and H280 both protonated at Nε. However, a neutral H250 with a proton at Nε and a cationic H280 may also be possible. Furthermore, the present QM/MM calculations of IR and Raman spectra of Agp1 containing isotope-labeled BV provide a detailed vibrational assignment of the biliverdin modes in the fingerprint region.
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