Mechanism of the Efficient Tryptophan Fluorescence Quenching in Human γD-Crystallin Studied by Time-Resolved Fluorescence†

Human γD-crystallin (HγD-Crys) is a two-domain, β-sheet eye lens protein found in the lens nucleus. Its long-term solubility and stability are important to maintain lens transparency throughout life. HγD-Crys has four highly conserved buried tryptophans (Trps), with two in each of the homologous β-sheet domains. In situ, these Trps will be absorbing ambient UV radiation that reaches the lens. The dispersal of the excited-state energy to avoid covalent damage is likely to be physiologically relevant for the lens crystallins. Trp fluorescence is efficiently quenched in native HγD-Crys. Previous steady-state fluorescence measurements provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain [Chen, J., et al. (2006) Biochemistry 45, 11552−11563]. Hybrid quantum mechanical−molecular mechanical (QM-MM) simulations indicated that the fluorescence of Trp68 and Trp156 is quenched by fast electron transfer to the amide backbone. Here we report additional information obtained using time-resolved fluorescence spectroscopy. In the single-Trp-containing proteins (Trp42-only, Trp68-only, Trp130-only, and Trp156-only), the highly quenched Trp68 and Trp156 have very short lifetimes, τ ~0.1 ns, whereas the moderately fluorescent Trp42 and Trp130 have longer lifetimes, τ ~3 ns. In the presence of the energy acceptor (Trp68 or Trp156), the lifetime of the energy donor (Trp42 or Trp130) decreased from ~3 to ~1 ns. The intradomain energy transfer efficiency is 56% in the N-terminal domain and is 71% in the C-terminal domain. The experimental values of energy transfer efficiency are in good agreement with those calculated theoretically. The absence of a time-dependent red shift in the time-resolved emission spectra of Trp130 proves that its local environment is very rigid. Time-resolved fluorescence anisotropy measurements with the single-Trp-containing proteins, Trp42-only and Trp130-only, indicate that the protein rotates as a rigid body and no segmental motion is detected. A combination of energy transfer with electron transfer results in short excited-state lifetimes of all Trps, which, together with the high rigidity of the protein matrix around Trps, could protect HγD-Crys from excited-state reactions causing permanent covalent damage

Mechanism of the efficient quenching of tryptophan fluorescence in human gamma crystallin

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008.Includes bibliographical references.Quenching of the fluorescence of buried tryptophans (Trps) is an important reporter of protein conformation. Human [gamma]D-crystallin (H[gamma]D-Crys) and human [gamma]S-crystallin (H[gamma]S-Crys) are both very stable eye lens protein that must remain soluble and folded throughout the human lifetime. Aggregation of non-native or covalently damaged H[gamma]D-Crys or H[gamma]S-Crys is associated with the prevalent eye disease mature-onset cataract. Both H[gamma]D-Crys and H[gamma]S-Crys have two homologous [beta]-sheet domains, each containing a pair of highly conserved buried tryptophans (see Fig. 1). The overall fluorescence of the Trps is quenched in the native state of H[gamma]D-Crys N-terminal domain C-terminal domain and H[gamma]S-Crys. In crystallin proteins, these Trps will Tr56 be absorbing UV radiation that reaches the lens. The dispersal of the excited state energy is likely to be H[gamma]siological relevant for the lens crystallins. Trp42 Steady-state and time-resolved fluorescence measurements combined with H[gamma]brid quantum Figure 1: The crystal structure of wild- mechanical-molecular mechanical (QM-MM) type H[gamma]D-Crys depicted in ribbon simulations revealed the quenching mechanism of representation showing the four H[gamma]D-Crys. From fluorescence of triple Trp to Phe intrinsic tryptophans in spacefill, mutants, the homologous pair Trp68 and Trpl56 are Trp42 and Trp68 in the N-terminal domain and Trpl30 and Trp156 in the found to be extremely quenched, with quantum C-terminal domain (Protein Data Bank yields close to 0.01, and with very short lifetimes, Code: 1HKO). T-0. 1ns. In contrast, the homologous pair Trp42 and Trpl30 are moderately fluorescent, with quantum yields of 0.13 and 0.17, respectively, and with longer lifetimes, T-3ns. In an attempt to identify quenching and/or electrostatically perturbing residues, a set of 17 candidate amino acids around Trp68 and Trp156 were substituted with neutral or H[gamma]drophobic residues.(cont.) None of these mutants showed significant changes in the fluorescence intensity compared to their own background. H[gamma]brid quantum mechanical-molecular mechanical (QM-MM) simulations were carried out by Prof. Patrik R. Callis at Montana State University. Computations with the four different excited Trps as electron donors strongly indicate that electron transfer rates to the amide backbone of Trp68 and Trp156 are extremely fast relative to those for Trp42 and Trpl30. This is in agreement with the quantum yields I measured experimentally and consistent with the absence of a quenching sidechain. Efficient electron transfer to the backbone is possible for Trp68 and Trp156 because of the net favorable location of several charged residues and the orientation of nearby waters, which collectively stabilize electron transfer electrostatically. The fluorescence emission spectra of single and double Trp to Phe mutants provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain. In the presence of the energy acceptor (Trp68 or Trp156), the lifetime of the energy donor (Trp42 or Trpl30) decreased from ~3ns to ~Ins. The intradomain energy transfer efficiency is 56% in the N-terminal domain and is 71% in the C-terminal domain. The experimental values of energy transfer efficiency are in good agreement with those calculated theoretically. Time-resolved fluorescence anisotropy measurements with the single-Trp containing proteins, Trp42-only and Trpl30-only, indicate that the protein rotates as a rigid body and no segmental motion is detected. The absence of a time-dependent red shift in the time-resolved emission spectra of Trpl30 proves that its local environment is very rigid.(cont.) A combination of energy transfer with electron transfer results in short excited-state lifetimes of all Trps, which, together with the high rigidity of the protein matrix around Trps, could protect H[gamma]D-Crys from excited-state reactions causing permanent covalent damage. Similar experimental and computational studies indicate that the quenching of the Trp fluorescence in H[gamma]S-Crys is also caused by fast electron transfer and intradomain Förster resonance energy transfer. The electrostatically enabled excited state quenching by electron transfer to the backbone amide is highly conserved in other [beta], [gamma]-crystallins despite the absence of precise sequence homology. This striking conservation, together with the observation of Tallmadge and Borkman [Tallmadge and Borkman, 1990] that the conserved quenched Trps in bovine [gamma]B-crystallin were protected from photolysis relative to the more fluorescent Trps, strongly suggests that the quenching is an evolved property of the protein fold that allows it to absorb ultraviolet light while suffering minimal photodamage.by Jiejin Chen.Ph.D

Mechanism of the Very Efficient Quenching of Tryptophan Fluorescence in Human γD- and γS-Crystallins: The γ-Crystallin Fold May Have Evolved To Protect Tryptophan Residues from Ultraviolet Photodamage

Proteins exposed to UV radiation are subject to irreversible photodamage through covalent modification of tryptophans (Trps) and other UV-absorbing amino acids. Crystallins, the major protein components of the vertebrate eye lens that maintain lens transparency, are exposed to ambient UV radiation throughout life. The duplicated β-sheet Greek key domains of β- and γ-crystallins in humans and all other vertebrates each have two conserved buried Trps. Experiments and computation showed that the fluorescence of these Trps in human γD-crystallin is very efficiently quenched in the native state by electrostatically enabled electron transfer to a backbone amide [Chen et al. (2006) <i>Biochemistry 45</i>, 11552−11563]. This dispersal of the excited state energy would be expected to minimize protein damage from covalent scission of the excited Trp ring. We report here both experiments and computation showing that the same fast electron transfer mechanism is operating in a different crystallin, human γS-crystallin. Examination of solved structures of other crystallins reveals that the Trp conformation, as well as favorably oriented bound waters, and the proximity of the backbone carbonyl oxygen of the n − 3 residues before the quenched Trps (residue n), are conserved in most crystallins. These results indicate that fast charge transfer quenching is an evolved property of this protein fold, probably protecting it from UV-induced photodamage. This UV resistance may have contributed to the selection of the Greek key fold as the major lens protein in all vertebrates

Age-related morphometrics of normal adrenal glands based on deep learning-aided segmentation

Objective.This study aims to evaluate the morphometrics of normal adrenal glands in adult patients semiautomatically using a deep learning-based segmentation model.Materials and Methods.A total of 520 abdominal CT image series with normal findings, from January 1, 2016, to March 14, 2019, were retrospectively collected for the training of the adrenal segmentation model. Then, 1043 portal venous phase image series of inpatient contrast-enhanced abdominal CT examinations with normal adrenal glands were included for analysis and grouped by every 10-year gap. A 3D U-Net-based segmentation model was used to predict bilateral adrenal labels followed by manual modification of labels as appropriate. Quantitative parameters (volume, CT value, and diameters) of the bilateral adrenal glands were then analyzed.Results.In the study cohort aged 18–77 years old (554 males and 489 females), the left adrenal gland was significantly larger than the right adrenal gland [all patients, 2867.79 (2317.11–3499.89) mm3 vs. 2452.84 (1983.50–2935.18) mm3, P < 0.001]. Male patients showed a greater volume of bilateral adrenal glands than females in all age groups (all patients, left: 3237.83 ± 930.21 mm3 vs. 2646.49 ± 766.42 mm3, P < 0.001; right: 2731.69 ± 789.19 mm3 vs. 2266.18 ± 632.97 mm3, P = 0.001). Bilateral adrenal volume in male patients showed an increasing then decreasing trend as age increased that peaked at 38–47 years old (left: 3416.01 ± 886.21 mm3, right: 2855.04 ± 774.57 mm3).Conclusions.The semiautomated measurement revealed that the adrenal volume differs as age increases. Male patients aged 38–47 years old have a peaked adrenal volume

Performance Study of Black Shale Modified Soil for Road Use Based on Eshelby–Mori–Tanaka Theory

Black shale, as a type of soft rock, exhibits high strength when freshly exposed. However, it easily disintegrates upon contact with water, making it unsuitable for direct use in roadbed construction. Using it as discarded material not only increases construction costs but also pollutes the environment. Therefore, the reuse and modification of black shale have become particularly important. Based on the theory of composite material equivalent inclusions, this study investigates the strength and water stability characteristics of black shale gravel after being mixed with cement and compacted with clay. The results show that the strength of cemented soil increases linearly with the cement content. The water absorption properties of the modified soils with different amounts of black shale added are similar, with an average water absorption rate of about 2.53%. The strength of black shale modified soil is generally positively correlated with the cement content, although the linear correlation is not significant. The modified black shale soil used in the experiment is suitable for the subgrade of medium- and light-grade secondary roads and below. The recommended mass ratio is Mshale:Mclay:Mcement = 70:21:9. The unconfined compressive strength of the material under 7-day curing is 1.36 MPa. The relationship between the strength of modified soil, clay strength, cement content, and gravel addition has been established, clarifying the physical significance of each parameter. The “drying and soaking” cycle can accelerate the strength degradation of modified soil. It is recommended to strengthen the construction of roadbed drainage facilities during construction to maintain a stable and dry environment for the modified soil as a roadbed filling material. The research results not only provide clear technical indicators for the reuse of discarded black shale in engineering but also serve as a basis for proportion of crushed stone discarded material as roadbed fill

Design, Synthesis and Biological Evaluation of Multi-Target Anti-Cancer Agent PYR26

This study investigates the synthesis of a new compound, PYR26, and the multi-target mechanism of PYR26 inhibiting the proliferation of HepG2 human hepatocellular carcinoma cells. PYR26 significantly inhibits the growth of HepG2 cells (p CDK4, c-Met and Bak genes in HepG2 cells were significantly inhibited (p p c-Met, CDK4 and Bak, up-regulating the mRNA expression of caspase-3 and Cyt c genes, down-regulating PI3K, pERK and CDK4 proteins and up-regulating the protein level of caspase-3. In a certain range, with the increase in PYR26 concentration, the tumor growth was slower and the tumor volume was smaller. Preliminary results showed that PYR26 also had an inhibitory effect on the tumors of Hepa1-6 tumor-bearing mice. These results suggest that PYR26 has an inhibitory effect on the growth of liver cancer cells, therefore it has potential to be developed into a new anti-liver cancer drug