Exploring the mycobacteriophage metaproteome: phage genomics as an educational platform

Bacteriophages are the most abundant forms of life in the biosphere and carry genomes characterized by high genetic diversity and mosaic architectures. The complete sequences of 30 mycobacteriophage genomes show them collectively to encode 101 tRNAs, three tmRNAs, and 3,357 proteins belonging to 1,536 “phamilies” of related sequences, and a statistical analysis predicts that these represent approximately 50% of the total number of phamilies in the mycobacteriophage population. These phamilies contain 2.19 proteins on average; more than half (774) of them contain just a single protein sequence. Only six phamilies have representatives in more than half of the 30 genomes, and only three—encoding tape-measure proteins, lysins, and minor tail proteins—are present in all 30 phages, although these phamilies are themselves highly modular, such that no single amino acid sequence element is present in all 30 mycobacteriophage genomes. Of the 1,536 phamilies, only 230 (15%) have amino acid sequence similarity to previously reported proteins, reflecting the enormous genetic diversity of the entire phage population. The abundance and diversity of phages, the simplicity of phage isolation, and the relatively small size of phage genomes support bacteriophage isolation and comparative genomic analysis as a highly suitable platform for discovery-based education.     

1,3-Dipolar cycloaddition of sugar azides with benzyne: a novel synthesis of 1,2,3-benzotriazolyl glycoconjugates

Glycosyl azides undergo smooth 1,3-dipolar cycloaddition with benzyne generated in situ from 2-(trimethylsilyl)phenyltrifluoromethanesulfonate and cesium fluoride under mild conditions to furnish 1,2,3-benzotriazole-linked glycoconjugates in excellent yields and with high stereoselectivity. This method provides a novel class of benzotriazole linked glycoconjugates in a single-step reaction. This is the first example of a fluoride- triggered 1,3-dipolar cycloaddition of benzyne with glycosyl azides.     

Organization of Valence-Encoding and Projection-Defined Neurons in the Basolateral Amygdala

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Rab9A is required for delivery of cargo from recycling endosomes to melanosomes

Melanosomes are a type of lysosome‐related organelle that is commonly defective in Hermansky–Pudlak syndrome. Biogenesis of melanosomes is regulated by BLOC‐1, ‐2, ‐3, or AP‐1, ‐3 complexes, which mediate cargo transport from recycling endosomes to melanosomes. Although several Rab GTPases have been shown to regulate these trafficking steps, the precise role of Rab9A remains unknown. Here, we found that a cohort of Rab9A associates with the melanosomes and its knockdown in melanocytes results in hypopigmented melanosomes due to mistargeting of melanosomal proteins to lysosomes. In addition, the Rab9A‐depletion phenotype resembles Rab38/32‐inactivated or BLOC‐3‐deficient melanocytes, suggesting that Rab9A works in line with BLOC‐3 and Rab38/32 during melanosome cargo transport. Furthermore, silencing of Rab9A, Rab38/32 or its effector VARP, or BLOC‐3‐deficiency in melanocytes decreased the length of STX13‐positive recycling endosomal tubules and targeted the SNARE to lysosomes. This result indicates a defect in directing recycling endosomal tubules to melanosomes. Thus, Rab9A and its co‐regulatory GTPases control STX13‐mediated cargo delivery to maturing melanosomes.     

Protein-Protein Docking with Dynamic Residue Protonation States

Protein-protein interactions depend on a host of environmental factors. Local pH conditions influence the interactions through the protonation states of the ionizable residues that can change upon binding. In this work, we present a pH-sensitive docking approach, pHDock, that can sample side-chain protonation states of five ionizable residues (Asp, Glu, His, Tyr, Lys) on-the-fly during the docking simulation. pHDock produces successful local docking funnels in approximately half (79/161) the protein complexes, including 19 cases where standard RosettaDock fails. pHDock also performs better than the two control cases comprising docking at pH 7.0 or using fixed, predetermined protonation states. On average, the top-ranked pHDock structures have lower interface RMSDs and recover more native interface residue-residue contacts and hydrogen bonds compared to RosettaDock. Addition of backbone flexibility using a computationally-generated conformational ensemble further improves native contact and hydrogen bond recovery in the top-ranked structures. Although pHDock is designed to improve docking, it also successfully predicts a large pH-dependent binding affinity change in the Fc–FcRn complex, suggesting that it can be exploited to improve affinity predictions. The approaches in the study contribute to the goal of structural simulations of whole-cell protein-protein interactions including all the environmental factors, and they can be further expanded for pH-sensitive protein design.     

Rapid Calculation of Protein pK(a) Values Using Rosetta

We developed a Rosetta-based Monte Carlo method to calculate the pK(a) values of protein residues that commonly exhibit variable protonation states (Asp, Glu, Lys, His, and Tyr). We tested the technique by calculating pK(a) values for 264 residues from 34 proteins. The standard Rosetta score function, which is independent of any environmental conditions, failed to capture pK(a) shifts. After incorporating a Coulomb electrostatic potential and optimizing the solvation reference energies for pK(a) calculations, we employed a method that allowed side-chain flexibility and achieved a root mean-square deviation (RMSD) of 0.83 from experimental values (0.68 after discounting 11 predictions with an error over 2 pH units). Additional degrees of side-chain conformational freedom for the proximal residues facilitated the capture of charge-charge interactions in a few cases, resulting in an overall RMSD of 0.85 pH units. The addition of backbone flexibility increased the overall RMSD to 0.93 pH units but improved relative pK(a) predictions for proximal catalytic residues. The method also captures large pK(a) shifts of lysine and some glutamate point mutations in staphylococcal nuclease. Thus, a simple and fast method based on the Rosetta score function and limited conformational sampling produces pK(a) values that will be useful when rapid estimation is essential, such as in docking, design, and folding.