Aromatic stacked salt bridges are increasingly observed to play an important role in biology, suggesting that the two separate weak interactions cooperate with each other to mediate molecular recognition in a biological solution. In this thesis an in depth study was carried out in attempt to find the contribution of the guanidinium-carboxylate-aromatic triad in biological systems. Two different small molecule systems are used to carry out the study. From the results of the two chapters I proposed here that stacking aromatic ring enhances the salt bridge through desolvation effect. This hypothesis was also tested in a protein-protein interaction (Grb2 SH3 domain/SOS interaction). The most ideal peptide inhibitor cannot be obtained due to the synthetic difficulties. Limited result showed that increasing the hydrophobic area of the hot spot in this protein-protein interaction enhances the interaction. In researching the guanidinium-carboxylate-aromatic triad, we were inspired to study the pre-organization effect of 1,3,5-triethyl-2,4,6-trisubstituted benzene template. A computational and literature study done in this thesis showed that the installation of ethyl or methyl groups at 1,3,5 positions leads to consistent increases in binding affinity relative to unsubstituted hosts, but the amount of increase is non-trivial and varies with different substitutes. The installation of ethyl or methyl groups at 1,3,5 positions leads to consistent but relatively small increases in binding affinity relative to unsubstituted hosts. / Graduate

This study investigated a CNF/dendrimer hydrogel and how different concentrations of the carboxylated CNF and bis-MPA ammonium dendrimer affected the hydrogels’ rheological properties. A third generation bis-MPA ammonium dendrimer was diffused into a dispersion of carboxylated cellulose nanofibrils. The CNF was carboxylated by TEMPO-oxidation and phosphate buffer deprotonating the carboxylic group. The ammonium dendrimers are cationic and, when added to the dispersion, act as a salt together with the CNF-carboxy anion creating a cationic dendrimer salt bridge. These will serve as physical crosslinks, and a CNF/dendrimer network is formed; the structure and the absorbed water make a hydrogel. Amplitude strain sweeps were performed with a rheometer to determine the gels' elastic capabilities in terms of storage modulus, G’ and loss modulus, G” as the function of the shear stress. The result shows that a higher concentration of both CNF dispersion and dendrimer yielded a higher value of the storage modulus and a lower critical strain, meaning that the hydrogel becomes firmer and less elastic. / I denna studie undersöktes en CNF/dendrimer hydrogel och hur olika koncentrationer av den karboxylerade CNF och bis-MPA ammonium dendrimer påverkar hydrogelens reologiska egenskaper. En tredje generations bis-MPA ammonium dendrimer läts diffusera i en dispersion av karboxylerade cellulosa nanofibriller (CNF). CNF karboxylerades via TEMPO-oxidation, varefter en fosfatbuffer adderades för att skapa en anjon. Dendrimerens ammoniumgrupper är katjoner och då den adderas till dispersionen kommer den agera som ett salt tillsammans med CNF-karboxyanjonen vilket skapar en katjonisk dendrimersaltbrygga. Denna agerar som en fysisk tvärbindning och skapar ett nätverk av CNF och dendrimer. Nätverket skapar tillsammans med det absorberade vattnet en hydrogel.  En amplitude strain sweep utfördes för att bestämma gelernas viskoelastiska förmåga, från mätningarna fås elasticitetsmodulen, G’ och den viskösa modulen, G’’ som funktioner av skjuvningen. Resultatet visar att en högre koncentration av CNF-dispersionen och dendrimeren leder till ett högre värde på elasticitetsmodulen samt ett lägre värde för den kritiska skjuvningen. Detta innebär att hydrogelen blir hårdare och mindre elastisk.

The structure of the voltage-gated proton (H+) channel Hv1 is homologous to the voltage sensor domain (VSD) of tetrameric voltage-gated Na+, K+ and Ca2+ channels (VGCs), but lacks a pore domain and instead forms a homodimer. Similar to other VSD proteins, Hv1 is gated by changes in membrane potential (V), but unlike VGCs, voltage-dependent gating in Hv1 is modulated by changes in the transmembrane pH gradient (DpH = pHo - pHi). In Hv1, pHo or pHi changes shift the open probability (POPEN)-V relation by ~40 mV per pH unit. To better understand the structural basis of pHo-dependent gating in Hv1, we constructed new resting- and activated-state Hv1 VSD homology models using physical constraints determined from experimental data measured under voltage clamp and conducted all-atom molecular dynamics (MD) simulations. Analyses of salt bridges and calculated pKas at conserved side chains suggests the existence of intracellular and extracellular electrostatic networks (ICEN and ECEN, respectively) that stabilize resting- or activated-state conformations of the Hv1 VSD. Structural analyses led to a novel hypothesis: two ECEN residues (E119 and D185) with coupled pKas coordinately interact with two S4 ‘gating charge’ Arg residues to modulate activated-state pHo sensitivity. Experimental data confirm that pH-dependent gating is compromised at acidic pHo in Hv1 E119A-D185A mutants, indicating that specific ECEN residue interactions are critical components of the ∆pH-dependent gating mechanism. E119 and D185 are known to participate in extracellular Zn2+ coordination, suggesting that H+ and Zn2+ utilize similar mechanisms to allosterically modulate the activated/resting state equilibrium in Hv1.

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.