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dc.rights.licenseRestricted to current Rensselaer faculty, staff and students. Access inquiries may be directed to the Rensselaer Libraries.
dc.contributorColón, Wilfredo
dc.contributorCramer, Steven M.
dc.contributorKarande, Pankaj
dc.contributorMakhatadze, George I.
dc.contributor.authorTrasatti, Hannah S.
dc.date.accessioned2021-11-03T09:01:09Z
dc.date.available2021-11-03T09:01:09Z
dc.date.created2018-07-27T15:11:48Z
dc.date.issued2018-05
dc.identifier.urihttps://hdl.handle.net/20.500.13015/2219
dc.descriptionMay 2018
dc.descriptionSchool of Science
dc.description.abstractStructural analysis of the thirteen kinetically stable proteins revealed common features among them. Understanding the structural basis of protein kinetic stability could allow prediction of this property from three dimensional protein structures, as well as inform the design of engineered proteins with high kinetic stability. Twelve of the thirteen proteins had either beta or combination alpha and beta (α+β fold) and nine were oligomeric. These structural properties have also been recognized in kinetically stable proteins from E. coli, V. cholerae, and V. harveyi. A novel computational analysis of the binding energy between the subunits of oligomeric kinetically stable proteins from human plasma and hemolysate was undertaken using the PRODIGY webserver. The results indicated that all the kinetically stable oligomers have strong interface energies that slow subunit dissociation. In addition, the topological complexity of the kinetically stable monomers and oligomer subunits was quantified using two calculations of contact order, a measure of the locality of residue-residue contacts. This type of analysis has not been performed on the subunits of oligomeric proteins before and could reveal additional protein kinetic stability, such as in the case of serum amyloid P component, that cannot be probed using D2D SDS PAGE. The results of the contact order calculations showed that these two parameters are useful in distinguishing monomeric proteins with high kinetic stability and low kinetic stability, but are ambiguous for cases in between. Further structural characteristics, such as void volume, electrostatic interactions, and ligand binding need to be incorporated to form a robust model for protein kinetic stability prediction.
dc.description.abstractProtein stability comes in two varieties: thermodynamic and kinetic stability. Thermodynamic protein stability is defined as the energy difference between the native and unfolded proteins states, while kinetic stability is defined as the energy difference between the native state and unfolding transition state. Proteins with high kinetic stability have slow unfolding rates and therefore, are resistant to denaturation by proteases and detergents. Protein thermodynamic stability is widely studied and its functional significance and pathological implications have been appreciated for decades. In contrast, the physical basis and functional significance of protein kinetic stability are still being elucidated. Protein kinetic stability has been linked to some protein misfolding diseases and may be relevant for other diseases as well. In this work, the subproteome of kinetically stable proteins and complexes in human plasma and red blood cells was explored using Diagonal 2 Dimensional (D2D) sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). This technique exploits the detergent resistance of kinetically stable proteins to identify them in biological samples.
dc.description.abstractBlood is a complex body fluid composed of plasma, platelets, red blood cells, and white blood cells. Human plasma is a unique diagnostic fluid that contains thousands of proteins and has great potential for biomarker discovery. D2D SDS PAGE analysis of human plasma followed by MALDI-TOF mass spectrometry and peptide mass fingerprinting led to the identification of ten non-redundant SDS-resistant proteins. Among them are six proteins that largely function in host defense (alpha 2 macroglobulin, complement C1r subcomponent, complement C3, complement C4, plasma protease C1 inhibitor, and serum amyloid P component) and four proteins involved in molecular transport (haptoglobin, retinol binding protein 4, serotransferrin, and transthyretin). Two of the kinetically stable proteins in plasma (plasma protease C1 inhibitor and transthyretin) are responsible for the development of disease when their kinetic stability is lost, demonstrating that change in a protein’s kinetic stability has the potential to be used as a novel diagnostic tool.
dc.description.abstractFrom a similar analysis of freshly prepared human hemolysate, three non-redundant proteins hemoglobin, catalase, and peroxiredoxin 2 were identified. Hemoglobin is a source of oxidative stress in the red blood cell via its iron ligands, whereas catalase and peroxiredoxin 2 are responsible for the elimination of dangerous reactive oxygen species as part of the antioxidant system. Protein kinetic stability may serve as a protective mechanism in these proteins to preserve their function by preventing misfolding after oxidative damage. Further depletion of human plasma and hemolysate is necessary to get a fuller picture of protein kinetic stability in humans. Due to the large protein abundance disparity in both samples, only a fraction of the proteins was probed for kinetic stability. Development of new methods to specifically deplete non kinetically stable protein out of complex biological samples would address this issue. Alternatively, a technique that is not limited by sample capacity would allow for examination of a broader range of proteins. Overall, the identification of thirteen kinetically stable proteins in human blood suggests that kinetic stability is an important characteristic that has been preserved throughout evolution, even as eukaryotes gained more complex protein function and regulation.
dc.language.isoENG
dc.publisherRensselaer Polytechnic Institute, Troy, NY
dc.relation.ispartofRensselaer Theses and Dissertations Online Collection
dc.subjectChemistry
dc.titleIdentification and analysis of kinetically stable proteins in human plasma and red blood cells
dc.typeElectronic thesis
dc.typeThesis
dc.digitool.pid179061
dc.digitool.pid179062
dc.digitool.pid179063
dc.rights.holderThis electronic version is a licensed copy owned by Rensselaer Polytechnic Institute, Troy, NY. Copyright of original work retained by author.
dc.description.degreePhD
dc.relation.departmentDept. of Chemistry and Chemical Biology


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