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Phosphorescence quenching studies of zinc substituted heme-proteins and novel applications of a water soluble hemicarcerand in biochemistry

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Title
Phosphorescence quenching studies of zinc substituted heme-proteins and novel applications of a water soluble hemicarcerand in biochemistry
Name (type = personal)
NamePart (type = family)
Jankowska
NamePart (type = given)
Katarzyna Izabela
NamePart (type = date)
1980-
DisplayForm
Katarzyna Jankowska
Role
RoleTerm (authority = RULIB)
author
Name (type = personal)
NamePart (type = family)
Sheridan
NamePart (type = given)
John
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John Sheridan
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Advisory Committee
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RoleTerm (authority = RULIB)
chair
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Piotrowiak
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Piotr
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Piotr Piotrowiak
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Advisory Committee
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internal member
Name (type = personal)
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Jordan
NamePart (type = given)
Frank
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Frank Jordan
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Advisory Committee
Role
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internal member
Name (type = personal)
NamePart (type = family)
Mendelsohn
NamePart (type = given)
Richard
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Richard Mendelsohn
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Advisory Committee
Role
RoleTerm (authority = RULIB)
internal member
Name (type = personal)
NamePart (type = family)
Warmuth
NamePart (type = given)
Ralf
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Ralf Warmuth
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
outside member
Name (type = corporate)
NamePart
Rutgers University
Role
RoleTerm (authority = RULIB)
degree grantor
Name (type = corporate)
NamePart
Graduate School - Newark
Role
RoleTerm (authority = RULIB)
school
TypeOfResource
Text
Genre (authority = marcgt)
theses
OriginInfo
DateCreated (qualifier = exact)
2012
DateOther (qualifier = exact); (type = degree)
2012-10
Place
PlaceTerm (type = code)
xx
Language
LanguageTerm (authority = ISO639-2b); (type = code)
eng
Subject (authority = RUETD)
Topic
Chemistry
Subject (authority = ETD-LCSH)
Topic
Hemoproteins
Subject (authority = ETD-LCSH)
Topic
Charge exchange
RelatedItem (type = host)
TitleInfo
Title
Rutgers University Electronic Theses and Dissertations
Identifier (type = RULIB)
ETD
Identifier
ETD_4164
PhysicalDescription
Form (authority = gmd)
electronic resource
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application/pdf
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text/xml
Extent
xxiii, 176 p. : ill.
Note (type = degree)
Ph.D.
Note (type = bibliography)
Includes bibliographical references
Note (type = vita)
Includes vita
Note (type = statement of responsibility)
by Katarzyna Izabela Jankowska
Subject (authority = ETD-LCSH)
Topic
Zinc proteins
Identifier (type = hdl)
http://hdl.rutgers.edu/1782.1/rucore10002600001.ETD.000066553
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TitleInfo
Title
Graduate School - Newark Electronic Theses and Dissertations
Identifier (type = local)
rucore10002600001
Location
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NjNbRU
Identifier (type = doi)
doi:10.7282/T3M907FH
Genre (authority = ExL-Esploro)
ETD doctoral
Abstract
Electron transfer (ET) reactions play a crucial role in biological systems. They occur at critical steps of numerous metabolic pathways and are studied in a variety of ways. One of the methods involves photoinduced ET investigation of chemically modified proteins, which are labeled or transformedto be photoactive. The most popular class of redox active proteins which allow modifications facilitating the study of ET reactions are heme proteins such as cytochromes. Their properties include colored prosthetic groups, varied oxidation states, and diverse biological functions which have provided a rich and fertile ground for study by chemists, biophysicists and biologists. The native iron-containing heme dissipates the excitation energy through rapid radiationless transitions which lead to extremely short excited state lifetimes in the femtosecond range. The replacement of iron by diamagnetic zinc at the porphyrin center dramatically changes the photophysics of the enzyme. Proteins containing zinc porphyrins exhibit both fluorescence and long lived phosphorescence Thanks to their emissive nature, they are amenable to highly sensitive single photon counting techniques, which permit kinetic studies over a very broad range of concentrations and time scales.

A large part of the research reported here has focused on examining intermolecular interactions in zinc substituted heme proteins probed by phosphorescence quenching. First, photoinduced ET reactions between zinc substituted cytochrome P450cam (ZnP450) and small organic compounds capable of accessing the protein’s hydrophobic channel and binding close to active site in a fashion that mimics its native substrate, camphor, were investigated.The heme – to - zinc protoporphyrin exchange revealed the existence of two conformers of the substituted protein (F420 and F450) which exhibited different photochemical and photophysical properties. The ET behavior of form F420 suggests that hydrophobic redox-active ligands are able to penetrate the hydrophobic channel and locate themselves in the direct vicinity of the Zn-porphyrin. In contrast, the slower ET quenching rates observed in the case of F450 indicate that the association is weak and occurs outside of the protein channel. Therefore, we conclude that form F420 corresponds to the open structure of the native cytochrome P450cam, while form F450 has a closed or partially closed channel that is characteristic of the camphor-containing cytochrome P450cam.

Both forms of ZnP450 were examined in the presence of ligands possessing long aliphaticchains to explore electron and energy transfer processes in those systems. The triplet state lifetime of form F420 decreases in all studied cases, while the emission of 3F450 remains unchanged in the presence of the selected ligands. The ET and TT rates obtained from 3F420 quenching allowed the calculation of the separation between the Zn-center and quenchers. The estimated D-A distances are consistent with those previously
obtained from X-ray structures of native cytochrome P450 and imply ligation close to the active center. Moreover, similarly as in native P450, quenchers with a hydrophobic tether induce conformational changes in ZnP450. These data show that ZnP450 mimics the behavior of the native enzyme and can be used to study protein - ligand and protein -protein interactions.

In the second part of this thesis a water soluble octacarboxyhemicarcerand was used as a shuttle to transport redox–active substrates across the aqueous medium and deliver them to the target protein. Hydrophobic electron donors and acceptors wereencapsulated within the hemicarcerand, and photoinduced electron transfer between the Zn-substitutedcytochrome c and the host-guest complexes was used to probe the association between the negatively charged hemicarceplex and the positively charged protein. ET mediated by the protein-bound hemicarcerand is much faster than that due to diffusional encounters with the respective free donor or acceptor in solution. The results show that the hemicarcerand is capable of exhibiting ‘induced fit’ behavior characteristic of protein-protein interactions. The kinetic behavior of these systems depends on the relative strength of the protein-hemicarcerand and guest-hemicarcerand interactions. When Kencaps >> Kassoc, the hemicarcerand transports the ligand to the protein while protecting it from the aqueous medium. But if Kassoc > Kencaps the docked cage can act as an artificial receptor.

In addition, the water soluble Cram-type hemicarcerand has been used as a gas capture device. This project involved encapsulation studies of hydrophobic gases such as sulfur hexafluoride or butane. The interactions between the guest and the host were probed by 1D relaxation and 2D NOESY experiments. The host:guest complex in a ratio of 1:1 was the dominant form for both studied gases, however additional NMR signals in the case of butane suggest imprisonment of two guests. The encapsulation of the second molecule is possible thanks to structural flexibility of a hemicarcerand, which can adapt to the guest. Furthermore, the fundamental question of the gas-like vs. liquid like behavior of the guest(s) in the inner phase environment of the hemicarcerand has been addressed. Our observations imply liquid like behavior for the 1:1 host: guest complex and solid-like behavior for the 1:2 host: butane complex, thus agreeing with Cram’s conjecture that the cage interior can be designed to be vacuum-like, liquid-like, or even solid-like depending on the fraction of the space occupied by the guest(s).

The presented work not only contributes new knowledge about photoinduced electron and energy transfer reactions in proteins but also introduces interesting materials for bio-sensing, biomedical or biocatalytic applications.
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The author owns the copyright to this work.
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Name
FamilyName
Jankowska
GivenName
Katarzyna
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DateTime (encoding = w3cdtf); (qualifier = exact); (point = start)
2012-06-26 11:26:26
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Katarzyna Jankowska
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Affiliation
Rutgers University. Graduate School - Newark
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I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
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DateTime (encoding = w3cdtf); (qualifier = exact); (point = start)
2012-10-31
DateTime (encoding = w3cdtf); (qualifier = exact); (point = end)
2013-10-31
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Access to this PDF has been restricted at the author's request. It will be publicly available after October 31st, 2013.
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