Biology subjects

Weaver, S. J.

Publications and source records attributed to Weaver, S. J..

6 recordsLinked to original sources

bioRxiv2025

Winter metabolic compensation endangers woodland salamanders under climate change

Understanding the mechanisms that shape species geographic distributions is essential for predicting responses to climate change, particularly under winter conditions, which are warming more rapidly than summer conditions in many temperate ecosystems. Woodland salamanders, which constitute many of the species in the global hotspot of salamander diversity located in the southern Appalachian Mountains of North America, are thought to cope with harsh winters through dormancy and metabolic suppression. We used a field-based experiment to investigate the winter physiology and energetics of a woodland salamander (Plethodon metcalfi). Despite showing histological signs of dormancy, salamanders exhibited higher metabolic rates during winter than the active season when measured at low temperatures associated with winter, consistent with metabolic compensation. High-elevation salamanders also exhibited elevated metabolic rates at cold temperatures compared to low-elevation salamanders, indicative of countergradient metabolic compensation. When integrated into a biophysical species distribution model, depletion of energy stores estimated from winter metabolic rates under current climates accurately predicted the geographic range limits of our focal species. Under future warming scenarios, incorporating winter physiology indicated that 77% of the species range would experience local extinction due to depletion of energy stores. These findings challenge assumptions about winter energetics, reveal metabolic limits on cold-season survival, and highlight metabolic demands during dormancy as a key constraint on responses to climate change.

ecology↗

bioRxiv2022

A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals

Crystallization of membrane proteins, such as G protein-coupled receptors (GPCRs), is challenging and frequently requires the use of lipidic cubic phase (LCP) crystallization methods. These typically yield crystals that are too small for synchrotron X-ray crystallography, but ideally suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, the viscous nature of LCP makes sample preparation challenging. The LCP layer is often too thick for transmission electron microscopy (TEM), and crystals buried in LCP cannot be identified topologically using a focused ion-beam and scanning electron microscope (FIB/SEM). Therefore, the LCP needs to either be converted to the sponge phase or entirely removed from the path of the ion-beam to allow identification and milling of these crystals. Unfortunately, conversion of the LCP to sponge phase can also deteriorate the sample. Methods that avoid LCP conversion are needed. Here, we employ a novel approach using an integrated fluorescence light microscope (iFLM) inside of a FIB/SEM to identify fluorescently labelled crystals embedded deep in a thick LCP layer. The crystals are then targeted using fluorescence microscopy and unconverted LCP is removed directly using a plasma focused ion beam (pFIB). To assess the optimal ion source to prepare biological lamellae, we first characterized the four available gas sources on standard crystals of the serine protease, proteinase K. However, lamellae prepared using either argon and xenon produced the highest quality data and structures. Fluorescently labelled crystals of the human adenosine receptor embedded in thick LCP were placed directly onto EM grids without conversion to the sponge phase. Buried microcrystals were identified using iFLM, and deep lamellae were created using the xenon beam. Continuous rotation MicroED data were collected from the exposed crystalline lamella and the structure was determined using a single crystal. This study outlines a robust approach to identifying and milling LCP grown membrane protein crystals for MicroED using single microcrystals, and demonstrates plasma ion-beam milling as a powerful tool for preparing biological lamellae.

biochemistry↗

bioRxiv2021

A faithful in vivo model of human macrophages in metastatic melanoma

AO_SCPLOWBSTRACTC_SCPLOWDespite recent therapeutic progress, advanced melanoma remains lethal for many patients. The composition of the immune tumor microenvironment (TME) has decisive impacts on therapy response and disease outcome. High dimensional analyses of patient samples can reveal the composition and heterogeneity of the immune TME. In particular, macrophages are known for their cancer-supportive role, but the underlying mechanisms are incompletely understood, and experimental in vivo systems are needed to test the functional properties of these cells. We characterized a humanized mouse model, reconstituted with a human immune system and a human melanoma, in which: (1) human macrophages support metastatic spread of the tumor; and (2) tumor-infiltrating macrophages have a specific transcriptional signature that faithfully represents the transcriptome of macrophages from patient melanoma samples and is associated with shorter survival. This model complements patient sample analyses, enabling the elucidation of fundamental principles in melanoma biology, and the development and evaluation of candidate therapies.

immunology↗

bioRxiv2020

Generation of ordered protein assemblies using rigid three-body fusion

Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A longstanding design approach uses genetic fusion to join protein homo-oligomer subunits via -helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method that performs rigid three-body fusion of homo-oligomer and spacer building blocks to generate user-defined architectures, while at the same time significantly increasing the number of geometric solutions over typical symmetric fusion. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies from a set of designed homo-dimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from DARPins (designed ankyrin repeat proteins), anchored on one end by -helical fusion and on the other by a designed homo-dimer interface, and we explored their use for cryo-EM structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects, small scaffold size, and the low-order symmetry of these dihedral scaffolds, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins.

synthetic biology↗

bioRxiv2020

CryoEM structure of the Vibrio cholerae Type IV competence pilus secretin PilQ

Natural transformation is the process by which bacteria take up genetic material from their environment and integrate it into their genome by homologous recombination. It represents one mode of horizontal gene transfer and contributes to the spread of traits like antibiotic resistance. In Vibrio cholerae, the Type IV competence pilus is thought to facilitate natural transformation by extending from the cell surface, binding to exogenous DNA, and retracting to thread this DNA through the outer membrane secretin, PilQ. A lack of structural information has hindered our understanding of this process, however. Here, we solved the first ever high-resolution structure of a Type IV competence pilus secretin. A functional tagged allele of VcPilQ purified from native V. cholerae cells was used to determine the cryoEM structure of the PilQ secretin in amphipol to [~]2.7 [A]. This structure highlights for the first time key differences in the architecture of the Type IV competence pilus secretin from the Type II and Type III Secretin System secretins. Based on our cryoEM structure, we designed a series of mutants to interrogate the mechanism of PilQ. These experiments provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation. We prove that it is possible to reduce pilus biogenesis and natural transformation by sealing the gate, suggesting VcPilQ as a new drug target.

biophysics↗

bioRxiv2018

Fusion of DARPin to aldolase enables visualization of small protein by cryoEM

In recent years, solving protein structures by single particle cryogenic electron microscopy (cryoEM) has become a crucial tool in structural biology. While exciting progress is being made towards the visualization of smaller and smaller macromolecules, the median protein size in both eukaryotes and bacteria is still beyond the reach of single particle cryoEM. To overcome this problem, we implemented a platform strategy in which a small protein target was rigidly attached to a large, symmetric base via a selectable adapter. Seven designs were tested. In the best construct, a designed ankyrin repeat protein (DARPin) was rigidly fused to tetrameric rabbit muscle aldolase through a helical linker. The DARPin retained its ability to bind its target, the 27 kDa green fluorescent protein (GFP). We solved the structure of this complex to 3.0 [A] resolution overall, with 5 to 8 [A] resolution in the GFP region. As flexibility in the DARPin limited the overall resolution of the target, we describe strategies to rigidify this element.\n\nAuthor summarySingle particle cryogenic electron microscopy (cryoEM) is a technique that uses images of purified proteins to determine their atomic structure. Unfortunately, the majority of proteins in the human and bacterial proteomes are too small to be analyzed by cryoEM. Over the years, several groups have suggested the use of a platform to increase the size of small protein targets. The platform is composed of a large protein base and a selectable adapter that binds the target protein. Here we report a platform based on tetrameric rabbit muscle aldolase that is fused to a Designed Ankyrin Repeat Protein (DARPin). Phage display libraries can be used to generate DARPins against target proteins. The residues mutated in a phage display library to generate a DARPin against a new target do not overlap with the DARPin-base fusion in the platform, thus changing the DARPin identity will not disrupt the platform design. The DARPin adapter used here is capable of binding Green Fluorescent Protein (GFP). We report the structure of GFP to 5 to 8 [A] local resolution by single particle cryoEM. Our analysis demonstrates that flexibility in the DARPin-aldolase platform prevents us from achieving higher resolution in the GFP region. We suggest changes to the DARPin design to rigidify the DARPin-aldolase platform. This work expands on current platforms and paves a generally applicable way toward structure determination of small proteins by cryoEM.

biophysics↗