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1. Glycine in Water Favors the Polyproline II State

   https://doi.org/10.3390/biom10081121  

   Andrews, Brian; Zhang, Shuting; Schweitzer-Stenner, Reinhard; Urbanc, Brigita (August 2020, Biomolecules)

   null (Ed.)

   Conformational preferences of amino acid residues in water are determined by the backbone and side-chain properties. Alanine is known for its high polyproline II (pPII) propensity. The question of relative contributions of the backbone and side chain to the conformational preferences of alanine and other amino acid residues in water is not fully resolved. Because glycine lacks a heavy-atom side chain, glycine-based peptides can be used to examine to which extent the backbone properties affect the conformational space. Here, we use published spectroscopic data for the central glycine residue of cationic triglycine in water to demonstrate that its conformational space is dominated by the pPII state. We assess three commonly used molecular dynamics (MD) force fields with respect to their ability to capture the conformational preferences of the central glycine residue in triglycine. We show that pPII is the mesostate that enables the functional backbone groups of the central residue to form the most hydrogen bonds with water. Our results indicate that the pPII propensity of the central glycine in GGG is comparable to that of alanine in GAG, implying that the water-backbone hydrogen bonding is responsible for the high pPII content of these residues. 

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2. Investigation of GTP-dependent dimerization of G12X K-Ras variants using ultraviolet photodissociation mass spectrometry

   https://doi.org/10.1039/c9sc01032g  

   Mehaffey, M. Rachel; Schardon, Christopher L.; Novelli, Elisa T.; Cammarata, Michael B.; Webb, Lauren J.; Fast, Walter; Brodbelt, Jennifer S. (August 2019, Chemical Science)

   Mutations in the GTPase enzyme K-Ras, specifically at codon G12, remain the most common genetic alterations in human cancers. The mechanisms governing activation of downstream signaling pathways and how they relate back to the identity of the mutation have yet to be completely defined. Here we use native mass spectrometry (MS) combined with ultraviolet photodissociation (UVPD) to investigate the impact of three G12X mutations (G12C, G12V, G12S) on the homodimerization of K-Ras as well as heterodimerization with a downstream effector protein, Raf. Electrospray ionization (ESI) was used to transfer complexes of WT or G12X K-Ras bound to guanosine 5′-diphosphate (GDP) or GppNHp (non-hydrolyzable analogue of GTP) into the gas phase. Relative abundances of homo- or hetero-dimer complexes were estimated from ESI-MS spectra. K-Ras + Raf heterocomplexes were activated with UVPD to probe structural changes responsible for observed differences in the amount of heterocomplex formed for each variant. Holo (ligand-bound) fragment ions resulting from photodissociation suggest the G12X mutants bind Raf along the expected effector binding region (β-interface) but may interact with Raf via an alternative α-interface as well. Variations in backbone cleavage efficiencies during UV photoactivation of each variant were used to relate mutation identity to structural changes that might impact downstream signaling. Specifically, oncogenic upregulation for hydrogen-bonding amino acid substitutions (G12C, G12S) is achieved by stabilizing β-interface interactions with Raf, while a bulkier, hydrophobic G12V substitution leads to destabilization of this interface and instead increases the proximity of residues along the α-helical bundles. This study deciphers new pieces of the complex puzzle of how different K-Ras mutations exert influence in downstream signaling. 

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3. Investigating Saccharomyces cerevisiae alkene reductase OYE 3 by substrate profiling, X-ray crystallography and computational methods

   https://doi.org/10.1039/c8cy00440d  

   Powell, III; Buteler, M. Pilar; Lenka, Sunidhi; Crotti, Michele; Santangelo, Sara; Burg, Matthew J.; Bruner, Steven; Brenna, Elisabetta; Roitberg, Adrian E.; Stewart, Jon D. (January 2018, Catalysis Science & Technology)

   Saccharomyces cerevisiae OYE 3 shares 80% sequence identity with the well-studied Saccharomyces pastorianus OYE 1; however, wild-type OYE 3 shows different stereoselectivities toward some alkene substrates. Site-saturation mutagenesis of Trp 116 in OYE 3 followed by substrate profiling showed that the mutations had relatively little effect, opposite to that observed previously for OYE 1. The X-ray crystal structures of unliganded and phenol-bound OYE 3 were solved to 1.8 and 1.9 Å resolution, respectively. Both structures were nearly identical to that of OYE 1, with only a single amino acid difference in the active site region (Ser 296 versus Phe 296, part of loop 6). Despite their essentially identical static X-ray structures, molecular dynamics (MD) simulations revealed that loop 6 conformations differed significantly in solution between OYE 3 and OYE 1. In OYE 3, loop 6 remained nearly as open as observed in the crystal structure; by contrast, loop 6 closed over the active site of OYE 1 by ca. 4 Å. Loop closure likely generates a greater number of active site protein contacts for substrate bound to OYE 1 as compared to OYE 3. These differences provide an explanation for the differing stereoselectivities of OYE 3 and OYE 1, despite their nearly identical X-ray crystal structures. 

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4. Acyl Capping Group Identity Effects on α-Helicity: On the Importance of Amide·Water Hydrogen Bonds to α-Helix Stability

   https://doi.org/10.1021/acs.biochem.3c00646  

   Bhatt, Megh R; Ganguly, Himal K; Zondlo, Neal J (May 2024, Biochemistry)

   Acyl capping groups stabilize -helices relative to free N-termini by providing one additional C=Oi•••Hi+4–N hydrogen bond. The electronic properties of acyl capping groups might also directly modulate -helix stability: electron-rich N-terminal acyl groups could stabilize the -helix by strengthening both i/i+4 hydrogen bonds and i/i+1 n* interactions. This hypothesis was tested in peptides X–AKAAAKAAAKAAAAKAAGY-NH2, X=different acyl groups. Surprisingly, the most electron-rich acyl groups (pivaloyl, iso-butyryl) strongly destabilized the -helix. Moreover, the formyl group induced nearly identical -helicity as the acetyl group, despite being a weaker electron donor for hydrogen bonds and for n* interactions. Other acyl groups exhibited intermediate -helicity. These results indicate that the electronic properties of the acyl carbonyl do not directly determine -helicity in peptides in water. In order to understand these effects, DFT calculations were conducted on -helical peptides. Using implicit solvation, -helix stability correlated with acyl group electronics, with the pivaloyl group exhibiting closer hydrogen bonds and n* interactions, in contrast to the experimental results. However, DFT and MD calculations with explicit water solvation revealed that hydrogen bonding to water was impacted by the sterics of the acyl capping group. Formyl capping groups exhibited the closest water-amide hydrogen bonds, while pivaloyl groups exhibited the longest. In -helices in the PDB, the highest frequency of close amide-water hydrogen bonds is observed when the N-cap residue is Gly. The combination of experimental and computational results indicates that solvation (hydrogen bonding of water) to the N-terminal amide groups is a central determinant of -helix stability. 

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5. An Investigation of the Effect of pH on Micelle Formation by a Glutamic Acid-Based Biosurfactant

   https://doi.org/10.3390/colloids8030038  

   Mayer, Jacob D; Rauscher, Robert M; Fritz, Shayden R; Fang, Yayin; Billiot, Eugene J; Billiot, Fereshteh H; Morris, Kevin F (June 2024, Colloids and Interfaces)

   NMR spectroscopy, molecular modeling, and conductivity experiments were used to investigate micelle formation by the amino acid-based surfactant tridecanoic L-glutamic acid. Amino acid-based biosurfactants are green alternatives to surfactants derived from petroleum. NMR titrations were used to measure the monomeric surfactant’s primary and gamma (γ) carboxylic acid pKa values. Intramolecular hydrogen bonding within the surfactant’s headgroup caused the primary carboxylic acid to be less acidic than the corresponding functional group in free L-glutamic acid. Likewise, intermolecular hydrogen bonding caused the micellar surfactant’s γ carboxylic functional group to be less acidic than the corresponding monomer value. The binding of four positive counterions to the anionic micelles was also investigated. At pH levels below 7.0 when the surfactant headgroup charge was −1, the micelle hydrodynamic radii were larger (~30 Å) and the mole fraction of micelle-bound counterions was in the 0.4–0.7 range. In the pH range of 7.0–10.5, the micelle radii decreased with increasing pH and the mole fraction of micelle bound counterions increased. These observations were attributed to changes in the surfactant headgroup charge with pH. Above pH 10.5, the counterions deprotonated and the mole fraction of micelle-bound counterions decreased further. Finally, critical micelle concentration measurements showed that the micelles formed at lower concentrations at pH 6 when the headgroup charge was predominately −1 and at higher concentrations at pH 7 where headgroups had a mixture of −1 and −2 charges in solution. 

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