Dissertations, Theses, and Capstone Projects

Date of Degree


Document Type


Degree Name





Ruel Z. B. Desamero

Committee Members

Brian R. Gibney

Richard S. Magliozzo

Sanjai K. Pathak

Adam A. Profit

Subject Categories

Biochemistry, Biophysics, and Structural Biology


amyloid fibrils, spectroscopy, peptide synthesis, aggregation, molecular dynamics


Amyloid fibril formation, the hallmark of numerous amyloid-related diseases, has been the subject of a vast number of scientific studies due to its pathological implications. Since the fibrillization process exhibits a certain level of intricacy, its investigation requires a multidisciplinary approach that integrates both experimental and computational methods. In vitro techniques involve biophysical assays and imaging tools for characterizing the structural and kinetic aspects of amyloid fibril formation. In parallel, in silico techniques offer programs for predicting atomistic details and behaviors of amyloidogenic proteins and peptides at the nanoscale level. Serum amyloid A (SAA), human islet amyloid polypeptide (hIAPP), and low-density lipoproteins (LDL) are examples of self-aggregating species whose accumulation has been associated with debilitating diseases. In this dissertation, both experimental and computational techniques were employed to gain comprehensive insights into the molecular mechanism governing their aggregation.

SAA is an acute-phase protein that is released during inflammation. Its accumulation and deposition of can lead to organ dysfunction and the progression of inflammatory diseases. Previous studies have proposed the N-terminal region of SAA to be its most amyloidogenic region, highlighting the significant roles of Arg-1, Ser-5, Glu-9, and Asp-12 in the fibrillization process. To test this, single-amino acid mutant peptides of the SAA1-13 fragment targeting these charged residues were synthesized and spectroscopically characterized. The influence of the mutations on the amyloidogenicity, accompanying secondary structural changes upon fibrillization, and fibril morphologies of the peptides were monitored and evaluated. Data revealed that mutating Arg-1, Ser-5, Glu-9, and Asp-12 resulted in the modulation of the amyloidogenic behavior and characteristics of the SAA1-13 peptide. Combined with MD simulation studies, it became possible to gain insight into the mechanistic implications of the molecular interactions formed by these key amino residues.

Amylin or hIAPP, a hormone co-secreted with insulin, is crucial in glucose homeostasis. However, its aggregation has been associated with the disease progression of type-2 diabetes by contributing to pancreatic β-cell dysfunction and death. A known aggregation-prone sequence of amylin includes residues 22-29 (hIAPP22-29), highlighting the importance of Phe-23 in the self-assembly process. Aromatic rings, such as phenylalanine and tryptophan, are known to interact with the arginine side chain via cation-π interactions. However, in some amyloidogenic proteins, it is the π-π stacking interactions formed by arginine residues that influence their amyloidogenicity. To test this, the effect of the guanidino group of arginine on the amyloidogenic propensity of NWGAILSS, an F23W mutant of hIAPP22-29, was investigated using non-resonance and UV resonance Raman (UVRR) spectroscopy. The mutation of Phe-23 to tryptophan was done since the tryptophan Raman modes are sensitive to different types of interactions. While the amyloidogenic propensity of NWGAILSS increased in the presence of Zn2+, co-incubating with the F23R mutant of hIAPP22-29 yielded the opposite result. The Zn2+-NWGAILSS interaction was attributable to cation-π contacts. The data collected along with the results of density functional theory (DFT)-based molecular simulation do not support a cation-π interaction between NRGAILSS and NWGAILSS. Instead, the in silico and in vitro results predicted π-π stacking between the guanidino group of NRGAILSS oriented parallel to the indole ring of NWGAILSS. Guanidino-group-containing peptide inhibitors of amylin amyloid formation were designed leveraging these unique interactions.

COVID-19 has been linked to an increased risk of developing type 2 diabetes (T2D), potentially exacerbated by preexisting metabolic syndrome and islet remodeling, with suggested mechanisms involving the aggregation of hIAPP. A previous computational study has proposed that the SARS-COV-2 peptide fragment SK9 (SFYVYSRVK) stabilizes the native conformation of hIAPP1-37 by interacting significantly with the N-terminal region of amylin, particularly stabilizing residues 15-28. Given these findings, we investigated whether SK9 could interact with short amyloidogenic sequences derived from this region, hIAPP12-18 and hIAPP20-29. Docking studies and simulations revealed that SK9 indeed stabilizes the helical conformation of hIAPP20-29. Moreover, SK9 inhibited the self-assembly of hIAPP20-29 in vitro, supporting the computational findings. Similar experiments were conducted with hIAPP12-18, showing SK9's ability to maintain helical conformation but to a greater extent. However, biophysical assays showed that SK9 does not prevent amyloid formation by full-length amylin. Thus, SK9 has no significant interaction with hIAPP1-37, contrary to previous computational predictions. These findings provide evidence contradicting previous computational models, elucidating potential mechanisms of SARS-COV-2 interaction with hIAPP and its relevance to T2D onset in COVID-19.

Self-aggregation of LDL is a known key factor in atherosclerotic cardiovascular disease. LDL’s electronegative form, LDL(-), possesses aggregation properties similar to amyloidogenic proteins. Studies suggest that its sole protein component, apolipoprotein B-100 (apo B-100), specifically the α2 domain, triggers LDL aggregation. Using secondary structure prediction tools, putative helical regions of the α2 domain of apo B-100 were modeled. MD simulations revealed that helices k (YFEKLVGFIDDAVK), m (YHQFVDETNDKIREVTQRLNGEIQA), and p (QQELQRYLSLVGQVYS) exhibited the formation of β-structures, a requirement for amyloid formation. Interactions such as π-π stacking between the aromatic rings seemed to drive the conformational transitions of the peptides. Helices k, m17-25 (QRLNGEIQA), and p were then experimentally synthesized and spectroscopically characterized. Based on the data, peptide p exhibited the highest amyloidogenicity, forming aggregates with structural characteristics similar to amyloid fibrils. Preliminary results also identified a modified peptide p fragment as a potential inhibitor against LDL aggregation.

Overall, this dissertation delves into the intricate process of aggregation of SAA, hIAPP, and LDL, through a combination of experimental and computational methods. The findings contribute to our understanding of amyloid formation and offer potential therapeutic strategies for preventing amyloid formation and its associated diseases.