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.
| Identifer | oai:union.ndltd.org:MIT/oai:dspace.mit.edu:1721.1/45428 |
| Date | January 2008 |
| Creators | Chen, Jiejin, Ph. D. Massachusetts Institute of Technology |
| Contributors | Jonathan King., Massachusetts Institute of Technology. Dept. of Chemistry., Massachusetts Institute of Technology. Dept. of Chemistry. |
| Publisher | Massachusetts Institute of Technology |
| Source Sets | M.I.T. Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 232 leaves, application/pdf |
| Rights | M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission., http://dspace.mit.edu/handle/1721.1/7582 |
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