With the nexus of super computing and the biotech revolution, it seems an era of predictive biology through systems biology may be at hand. Modern omics capabilities enable examination of the state ...of biological system in exquisite detail. The genome, transcriptome, proteome, and metabolome may all be largely knowable, at least for some model systems, providing a basis for modeling and simulation of molecular mechanisms, or pathways, that could capture a biological system's emergent properties. However, there are significant challenges remaining that impede the realization of this vision, perhaps the most significant being the missing functional annotation of genes and gene products. For even the most well-studied organisms as much as a third of called genes for a given genome are not annotated and more than half may be tenuous. Homology inferred from sequence similarity is the basis for much of genome annotation. Homology inferred from structural similarity could be a powerful complement to sequence-based annotation methods. Structural biology or structural informatics can be used to assign molecular function and may have increasing utility with the rapid growth of gene sequence databases and emerging methods for structure determination, like structure prediction based on coevolution. Here we describe tools and provide example cases using structural similarity at the level of quaternary structure, domain content, domain topology, and small 3D motifs to infer homology and posit function. Ultimately annotation by similarity, be it 3D structure homology or more classically primary sequence homology, must be founded by accurate annotation of one ortholog in the group-understanding every function encoded by a genome remains a major challenge to life science.
Abstract
Alchemical free energy perturbation (FEP) is a rigorous and powerful technique to calculate the free energy difference between distinct chemical systems. Here we report our implementation of ...automated large-scale FEP calculations, using the Amber software package, to facilitate antibody design and evaluation. In combination with Hamiltonian replica exchange, our FEP simulations aim to predict the effect of mutations on both the binding affinity and the structural stability. Importantly, we incorporate multiple strategies to faithfully estimate the statistical uncertainties in the FEP results. As a case study, we apply our protocols to systematically evaluate variants of the m396 antibody for their conformational stability and their binding affinity to the spike proteins of SARS-CoV-1 and SARS-CoV-2. By properly adjusting relevant parameters, the particle collapse problems in the FEP simulations are avoided. Furthermore, large statistical errors in a small fraction of the FEP calculations are effectively reduced by extending the sampling, such that acceptable statistical uncertainties are achieved for the vast majority of the cases with a modest total computational cost. Finally, our predicted conformational stability for the m396 variants is qualitatively consistent with the experimentally measured melting temperatures. Our work thus demonstrates the applicability of FEP in computational antibody design.
Peptide-based subunit vaccines are coming to the forefront of current vaccine approaches, with safety and cost-effective production among their top advantages. Peptide vaccine formulations consist of ...multiple synthetic linear epitopes that together trigger desired immune responses that can result in robust immune memory. The advantages of linear compared to conformational epitopes are their simple structure, ease of synthesis, and ability to stimulate immune responses by means that do not require complex 3D conformation. Prediction of linear epitopes through use of computational tools is fast and cost-effective, but typically of low accuracy, necessitating extensive experimentation to verify results. On the other hand, identification of linear epitopes through experimental screening has been an inefficient process that requires thorough characterization of previously identified full-length protein antigens, or laborious techniques involving genetic manipulation of organisms. In this study, we apply a newly developed generalizable screening method that enables efficient identification of B-cell epitopes in the proteomes of pathogenic bacteria. As a test case, we used this method to identify epitopes in the proteome of
(Ft), a Select Agent with a well-characterized immunoproteome. Our screen identified many peptides that map to known antigens, including verified and predicted outer membrane proteins and extracellular proteins, validating the utility of this approach. We then used the method to identify seroreactive peptides in the less characterized immunoproteome of Select Agent
(Bp). This screen revealed known Bp antigens as well as proteins that have not been previously identified as antigens. Although B-cell epitope prediction tools Bepipred 2.0 and iBCE-EL classified many of our seroreactive peptides as epitopes, they did not score them significantly higher than the non-reactive tryptic peptides in our study, nor did they assign higher scores to seroreactive peptides from known Ft or Bp antigens, highlighting the need for experimental data instead of relying on computational epitope predictions alone. The present workflow is easily adaptable to detecting peptide targets relevant to the immune systems of other mammalian species, including humans (depending upon the availability of convalescent sera from patients), and could aid in accelerating the discovery of B-cell epitopes and development of vaccines to counter emerging biological threats.
The polymorphic membrane proteins (Pmps) are a family of autotransporters that play an important role in infection, adhesion and immunity in Chlamydia trachomatis. Here we show that the ...characteristic GGA(I,L,V) and FxxN tetrapeptide repeats fit into a larger repeat sequence, which correspond to the coils of a large beta-helical domain in high quality structure predictions. Analysis of the protein using structure prediction algorithms provided novel insight to the chlamydial Pmp family of proteins. While the tetrapeptide motifs themselves are predicted to play a structural role in folding and close stacking of the beta-helical backbone of the passenger domain, we found many of the interesting features of Pmps are localized to the side loops jutting out from the beta helix including protease cleavage, host cell adhesion, and B-cell epitopes; while T-cell epitopes are predominantly found in the beta-helix itself. This analysis more accurately defines the Pmp family of Chlamydia and may better inform rational vaccine design and functional studies.
and
are the causative agents of melioidosis and glanders, respectively, and are often fatal to humans and animals. Owing to the high fatality rate, potential for spread by aerosolization, and the ...lack of efficacious therapeutics,
and
are considered biothreat agents of concern. In this study, we investigate the proteome of
, a closely related surrogate for the two more virulent
species, during infection of host cells, and compare to that of
in culture. Studying the proteome of
spp. during infection is expected to reveal molecular mechanisms of intracellular survival and host immune evasion; but proteomic profiling of
during host infection is challenging. Proteomic analyses of host-associated bacteria are typically hindered by the overwhelming host protein content recovered from infected cultures. To address this problem, we have applied bio-orthogonal noncanonical amino acid tagging (BONCAT) to
, enabling the enrichment of newly expressed bacterial proteins from virtually any growth condition, including host cell infection. In this study, we show that
proteins were selectively labeled and efficiently enriched from infected host cells using BONCAT. We also demonstrate that this method can be used to label bacteria
by fluorescent tagging. Finally, we present a global proteomic profile of
as it infects host cells and a list of proteins that are differentially regulated in infection conditions as compared to bacterial monoculture. Among the identified proteins are quorum sensing regulated genes as well as homologs to previously identified virulence factors. This method provides a powerful tool to study the molecular processes during
infection, a much-needed addition to the
molecular toolbox.
Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading ...to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Δ1–49 apolipoprotein A-I fragment (Δ49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR → M transition. Importantly the functional bR was solubilized in discoidal bR·NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Δ49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies.
Legionella
is a genus of ubiquitous environmental pathogens found in freshwater systems, moist soil, and composted materials. More than four decades of
Legionella
research has provided important ...insights into
Legionella
pathogenesis. Although standard commercial microscopes have led to significant advances in understanding
Legionella
pathogenesis, great potential exists in the deployment of more advanced imaging techniques to provide additional insights. The lattice light sheet microscope (LLSM) is a recently developed microscope for 4D live cell imaging with high resolution and minimum photo-damage. We built a LLSM with an improved version for the optical layout with two path-stretching mirror sets and a novel reconfigurable galvanometer scanner (
RGS
) module to improve the reproducibility and reliability of the alignment and maintenance of the LLSM. We commissioned this LLSM to study
Legionella pneumophila
infection with a tailored workflow designed over instrumentation, experiments, and data processing methods. Our results indicate that
Legionella pneumophila
infection is correlated with a series of morphological signatures such as smoothness, migration pattern and polarity both statistically and dynamically. Our work demonstrates the benefits of using LLSM for studying long-term questions in bacterial infection. Our free-for-use modifications and workflow designs on the use of LLSM system contributes to the adoption and promotion of the state-of-the-art LLSM technology for both academic and commercial applications.
X‐ray free‐electron lasers (XFELs) offer a new avenue to the structural probing of complex materials, including biomolecules. Delivery of precious sample to the XFEL beam is a key consideration, as ...the sample of interest must be serially replaced after each destructive pulse. The fixed‐target approach to sample delivery involves depositing samples on a thin‐film support and subsequent serial introduction via a translating stage. Some classes of biological materials, including two‐dimensional protein crystals, must be introduced on fixed‐target supports, as they require a flat surface to prevent sample wrinkling. A series of wafer and transmission electron microscopy (TEM)‐style grid supports constructed of low‐Z plastic have been custom‐designed and produced. Aluminium TEM grid holders were engineered, capable of delivering up to 20 different conventional or plastic TEM grids using fixed‐target stages available at the Linac Coherent Light Source (LCLS). As proof‐of‐principle, X‐ray diffraction has been demonstrated from two‐dimensional crystals of bacteriorhodopsin and three‐dimensional crystals of anthrax toxin protective antigen mounted on these supports at the LCLS. The benefits and limitations of these low‐Z fixed‐target supports are discussed; it is the authors' belief that they represent a viable and efficient alternative to previously reported fixed‐target supports for conducting diffraction studies with XFELs.
Spontaneous interaction of purified apolipoproteins and phospholipids results in formation of lipoprotein particles with nanometer-sized dimensions; we refer to these assemblies as nanolipoprotein ...particles or NLPs. These bilayer constructs can serve as suitable mimetics of biological membranes and are fully soluble in aqueous environments. We made NLPs from dimyristoylphospatidylcholine (DMPC) in combination with each of four different apolipoproteins: apoA-I, Δ-apoA-I fragment, apoE4 fragment, and apolipophorin III (apoLp-III) from the silk moth B. mori. Predominately discoidal in shape, these particles have diameters between 10 and 20 nm, share uniform heights between 4.5 and 5 nm, and can be produced in yields ranging between 40 and 60%. The particular lipoprotein, the lipid to lipoprotein ratio, and the assembly parameters determine the size and homogeneity of nanolipoprotein particles and indicate that apoA-I NLP preparations are smaller than the larger apoE422K and apoLp-III NLP preparations.
Nanolipoprotein particles (NLPs) are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins. NLPs assembled with human apolipoproteins have been ...used for myriad biotechnology applications, including membrane protein solubilization, drug delivery, and diagnostic imaging. To expand the repertoire of lipoproteins for these applications, insect apolipophorin-III (apoLp-III) was evaluated for the ability to form discretely-sized, homogeneous, and stable NLPs.
Four NLP populations distinct with regards to particle diameters (ranging in size from 10 nm to >25 nm) and lipid-to-apoLp-III ratios were readily isolated to high purity by size exclusion chromatography. Remodeling of the purified NLP species over time at 4 degrees C was monitored by native gel electrophoresis, size exclusion chromatography, and atomic force microscopy. Purified 20 nm NLPs displayed no remodeling and remained stable for over 1 year. Purified NLPs with 10 nm and 15 nm diameters ultimately remodeled into 20 nm NLPs over a period of months. Intra-particle chemical cross-linking of apoLp-III stabilized NLPs of all sizes.
ApoLp-III-based NLPs can be readily prepared, purified, characterized, and stabilized, suggesting their utility for biotechnological applications.
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