PEGylation can improve the pharmacokinetic properties of protein therapeutics via decreasing renal clearance and shielding the protein surface from proteases, antibody neutrailization, and aggregation. Conformational stability enhancement can provide criteria for the identification of optimal sites for PEGylation, but how PEG influence the noncovalent interactions from the surface of proteins has not been well illustrated. Macrocyclization can effectively enhance the conformational stability of small peptides and large proteins. Combination of PEG-based conformational stability enhancement and macrocyclization-based conformational constraint has not been explored. Macrocycliziation has been employed to stabilize protein tertiary structures, but there are no general guidelines for interhelical staple to stabilize coiled-coil motifs of proteins. Chapter 1 is an introduction to peptide stapling and macrocyclization of proteins. Chapter 2 describes our test of the hypothesis that PEG increases the conformational stability of proteins by desolvating nearby salt bridges. In chapter 3, we explore the combination of PEG-based conformational stability enhancement with macrocyclization on WW domain, and find that the most important criteria for PEG stapling is ensuring the side chains cross-linked by PEG are distant in primary sequence but close in tertiary structure. In chapter 4, we further apply this macrocyclization criteria to another -sheet-based protein, SH3 domain of the chicken Src protein, and to a disulfide-bonded parallel coiled-coil heterodimer derived from the yeast transcription factor GCN4. In chapter 5, we explore the determinants of PEG-staple-based stabilization by changing the distance of the staple to the terminal interhelical disulfide bond, varying the length of staple, exploring different solvent exposed positions for stapling and employing heterochiral residues for stapling. We further apply the interhelical PEG staple to a HER-2 affibody, and find that PEG-stapling increases the conformational stability and proteolytic resistance of the stapled affibody relative to its non-stapled counterpart and to the native unmodified affibody.