AlphaFold-assisted structure determination of a bacterial protein of unknown function using X-ray and electron crystallography.

Macromolecular crystallography generally requires the recovery of missing phase information from diffraction data to reconstruct an electron-density map of the crystallized molecule. Most recent structures have been solved using molecular replacement as a phasing method, requiring an a priori structure that is closely related to the target protein to serve as a search model; when no such search model exists, molecular replacement is not possible. New advances in computational machine-learning methods, however, have resulted in major advances in protein structure predictions from sequence information. Methods that generate predicted structural models of sufficient accuracy provide a powerful approach to molecular replacement. Taking advantage of these advances, AlphaFold predictions were applied to enable structure determination of a bacterial protein of unknown function (UniProtKB Q63NT7, NCBI locus BPSS0212) based on diffraction data that had evaded phasing attempts using MIR and anomalous scattering methods. Using both X-ray and micro-electron (microED) diffraction data, it was possible to solve the structure of the main fragment of the protein using a predicted model of that domain as a starting point. The use of predicted structural models importantly expands the promise of electron diffraction, where structure determination relies critically on molecular replacement.

Design of Ligand-Operable Protein-Cages That Open Upon Specific Protein Binding.

Protein nanocages have diverse applications in medicine and biotechnology, including molecular delivery. However, although numerous studies have demonstrated the ability of protein nanocages to encapsulate various molecular species, limited methods are available for subsequently opening a nanocage for cargo release under specific conditions. A modular platform with a specific protein-target-based mechanism of nanocage opening is notably lacking. To address this important technology gap, we present a new class of designed protein cages, the Ligand-Operable Cage (LOC). LOCs primarily comprise a protein nanocage core and a fused surface binding adaptor. The geometry of the LOC is designed so that binding of a target protein ligand (or multiple copies thereof) to the surface binder is sterically incompatible with retention of the assembled state of the cage. Therefore, the tight binding of a target ligand drives cage disassembly by mass action, subsequently exposing the encapsulated cargo. LOCs are modular; direct substitution of the surface binder sequence can reprogram the nanocage to open in response to any target protein ligand of interest. We demonstrate these design principles using both a natural and a designed protein cage as the core, with different proteins acting as the triggering ligand and with different reporter readouts─fluorescence unquenching and luminescence─for cage disassembly. These developments advance the critical problem of targeted molecular delivery and detection.

Design of Beta-2 Microglobulin Adsorbent Protein Nanoparticles.

Beta-2 microglobulin (B2M) is an immune system protein that is found on the surface of all nucleated human cells. B2M is naturally shed from cell surfaces into the plasma, followed by renal excretion. In patients with impaired renal function, B2M will accumulate in organs and tissues leading to significantly reduced life expectancy and quality of life. While current hemodialysis methods have been successful in managing electrolyte as well as small and large molecule disturbances arising in chronic renal failure, they have shown only modest success in managing plasma levels of B2M and similar sized proteins, while sparing important proteins such as albumin. We describe a systematic protein design effort aimed at adding the ability to selectively remove specific, undesired waste proteins such as B2M from the plasma of chronic renal failure patients. A novel nanoparticle built using a tetrahedral protein assembly as a scaffold that presents 12 copies of a B2M-binding nanobody is described. The designed nanoparticle binds specifically to B2M through protein-protein interactions with nanomolar binding affinity (~4.2 nM). Notably, binding to the nanoparticle increases the effective size of B2M by over 50-fold, offering a potential selective avenue for separation based on size. We present data to support the potential utility of such a nanoparticle for removing B2M from plasma by either size-based filtration or by polyvalent binding to a stationary matrix under blood flow conditions. Such applications could address current shortcomings in the management of problematic mid-sized proteins in chronic renal failure patients.

Evaluating the active site-substrate interplay between x-ray crystal structure and molecular dynamics in chorismate mutase.

Designing realistic quantum mechanical (QM) models of enzymes is dependent on reliably discerning and modeling residues, solvents, and cofactors important in crafting the active site microenvironment. Interatomic van der Waals contacts have previously demonstrated usefulness toward designing QM-models, but their measured values (and subsequent residue importance rankings) are expected to be influenceable by subtle changes in protein structure. Using chorismate mutase as a case study, this work examines the differences in ligand-residue interatomic contacts between an x-ray crystal structure and structures from a molecular dynamics simulation. Select structures are further analyzed using symmetry adapted perturbation theory to compute ab initio ligand-residue interaction energies. The findings of this study show that ligand-residue interatomic contacts measured for an x-ray crystal structure are not predictive of active site contacts from a sampling of molecular dynamics frames. In addition, the variability in interatomic contacts among structures is not correlated with variability in interaction energies. However, the results spotlight using interaction energies to characterize and rank residue importance in future computational enzymology workflows.

Designing Protease-Triggered Protein Cages.

Proteins that self-assemble into enclosed polyhedral cages, both naturally and by design, are garnering attention for their prospective utility in the fields of medicine and biotechnology. Notably, their potential for encapsulation and surface display are attractive for experiments that require protection and targeted delivery of cargo. The ability to control their opening or disassembly would greatly advance the development of protein nanocages into widespread molecular tools. Toward the development of protein cages that disassemble in a systematic manner and in response to biologically relevant stimuli, here we demonstrate a modular protein cage system that is opened by highly sequence-specific proteases, based on sequence insertions at strategically chosen loop positions in the protein cage subunits. We probed the generality of the approach in the context of protein cages built using the two prevailing methods of construction: genetic fusion between oligomeric components and (non-covalent) computational interface design between oligomeric components. Our results suggest that the former type of cage may be more amenable than the latter for endowing proteolytically controlled disassembly. We show that a successfully designed cage system, based on oligomeric fusion, is modular with regard to its triggering protease. One version of the cage is targeted by an asparagine protease implicated in cancer and Alzheimer's disease, whereas the second version is responsive to the blood-clotting protease, thrombin. The approach demonstrated here should guide future efforts to develop therapeutic vectors to treat disease states where protease induction or mis-regulation occurs.

Abnormal development of cerebral arteries and veins in offspring of experimentally preeclamptic rats: Potential role in perinatal stroke.

Preeclampsia, a hypertensive disorder of pregnancy, complicates up to 10 % of all pregnancies and increases the risk for perinatal stroke in offspring. The mechanism of this increase is unknown, but may involve vascular dysfunction. The goal of this study was to evaluate the effect of experimental preeclampsia (ePE) on cerebrovascular function in offspring to eludciate a possible mechanism for this association. Dams were fed a high cholesterol diet beginning on day 7 of gestation to induce experimental preeclampsia. Middle cerebral arteries (MCA) and the Vein of Galen (VoG) were isolated from pups from ePE dams and compared to pups from normal pregnant (NP) dams at postnatal days 16, 23, and 30 and studied pressurized in an arteriograph chamber. Markers of inflammation and oxidative stress were measured in serum. Our results suggest altered structure and function in both MCA and VoG of ePE pups. We also found evidence of systemic inflammation and oxidative stress in ePE pups. These findings provide a potential link between preeclampsia and the occurrence or severity of perinatal stroke.

Geometric Lessons and Design Strategies for Nanoscale Protein Cages.

Protein molecules bring a rich functionality to the field of designed nanoscale architectures. High-symmetry protein cages are rapidly finding diverse applications in biomedicine, nanotechnology, and imaging, but methods for their reliable and predictable construction remain challenging. In this study we introduce an approach for designing protein assemblies that combines ideas and favorable elements adapted from recent work. Cubically symmetric cages can be created by combining two simpler symmetries, following recently established principles. Here, two different oligomeric protein components are brought together in a geometrically specific arrangement by their separate genetic fusion to individual components of a heterodimeric coiled-coil polypeptide motif of known structure. Fusions between components are made by continuous α-helices to limit flexibility. After a computational design, we tested 10 different protein cage constructions experimentally, two of which formed larger assemblies. One produced the intended octahedral cage, ∼26 nm in diameter, while the other appeared to produce the intended tetrahedral cage as a minor component, crystallizing instead in an alternate form representing a collapsed structure of lower stoichiometry and symmetry. Geometric distinctions between the two characterized designs help explain the different degrees of success, leading to clearer principles and improved prospects for the routine creation of nanoscale protein architectures using diverse methods.

Memory impairment in spontaneously hypertensive rats is associated with hippocampal hypoperfusion and hippocampal vascular dysfunction.

We investigated the effect of chronic hypertension on hippocampal arterioles (HippAs) and hippocampal perfusion as underlying mechanisms of memory impairment, and how large artery stiffness relates to HippA remodeling. Using male spontaneously hypertensive rats (SHR) and normotensive Wistar rats (n = 12/group), long-term (LTM) and spatial memory were tested using object recognition and spontaneous alternation tasks. Hippocampal blood flow was measured via hydrogen clearance basally and during hypercapnia. Reactivity of isolated and pressurized HippAs to pressure and pharmacological activators and inhibitors was investigated. To determine large artery stiffness, distensibility and elastin content were measured in thoracic aorta. SHR had impaired LTM and spatial memory associated with decreased basal blood flow (68 ± 12 mL/100 g/min) vs. Wistar (111 ± 28 mL/100 g/min, p < 0.01) that increased during hypercapnia similarly between groups. Compared to Wistar, HippAs from SHR had increased tone at 60 mmHg (58 ± 9% vs. 37 ± 7%, p < 0.01), and decreased reactivity to small- and intermediate-conductance calcium-activated potassium (SK/IK) channel activation. HippAs in both groups were unaffected by NOS inhibition. Decreased elastin content correlated with increased stiffness in aorta of SHR that was associated with increased stiffness and hypertrophic remodeling of HippAs. Hippocampal vascular dysfunction during hypertension could potentiate memory deficits and may provide a therapeutic target to limit vascular cognitive impairment.

Genome Sequences of Cluster K Mycobacteriophages Deby, LaterM, LilPharaoh, Paola, SgtBeansprout, and Sulley.

Mycobacteriophages Deby, LaterM, LilPharaoh, Paola, SgtBeansprout, and Sulley were isolated from soil using Mycobacterium smegmatis mc2155. Genomic analysis indicated that they belong to subclusters K1 and K5. Their genomic architectures are typical of cluster K mycobacteriophages, with most variability occurring on the right end of the genome sequence.

The Antibiotic Trimethoprim Displays Strong Mutagenic Synergy with 2-Aminopurine.

We show that trimethoprim (TMP), an antibiotic in current use, displays a strong synergistic effect on mutagenesis in Escherichia coli when paired with the base analog 2-aminopurine (2AP), resulting in a 35-fold increase in mutation frequencies in the rpoB-Rifr system. Combination therapies are often employed both as antibiotic treatments and in cancer chemotherapy. However, mutagenic effects of these combinations are rarely examined. An analysis of the mutational spectra of TMP, 2AP, and their combination indicates that together they trigger a response via an alteration in deoxynucleoside triphosphate (dNTP) ratios that neither compound alone can trigger. A similar, although less strong, response is seen with the frameshift mutagen ICR191 and 2AP. These results underscore the need for testing the effects on mutagenesis of combinations of antibiotics and chemotherapeutics.