Marian Baclayon

Norwalk Virus Assembly and Stability Monitored by Mass Spectrometry

Viral capsid assembly, in which viral proteins self-assemble into complexes of well defined architecture, is a fascinating biological process. Although viral structure and assembly processes have been the subject of many excellent structural biology studies in the past, questions still remain regarding the intricate mechanisms that underlie viral structure, stability, and assembly. Here we used native mass spectrometry-based techniques to study the structure, stability, and assembly of Norwalk virus-like particles. Although detailed structural information on the fully assembled capsid exists, less information is available on potential capsid (dis)assembly intermediates, largely because of the inherent heterogeneity and complexity of the disassembly pathways. We used native mass spectrometry and atomic force microscopy to investigate the (dis)assembly of the Norwalk virus-like particles as a function of solution pH, ionic strength, and VP1 protein concentration. Native MS analysis at physiological pH revealed the presence of the complete capsid (T = 3) consisting of 180 copies of VP1. The mass of these capsid particles extends over 10 million Da, ranking them among the largest protein complexes ever analyzed by native MS. Although very stable under acidic conditions, the capsid was found to be sensitive to alkaline treatment. At elevated pH, intermediate structures consisting of 2, 4, 6, 18, 40, 60, and 80 copies of VP1 were observed with the VP160 (3.36-MDa) and VP180 (4.48-MDa) species being most abundant. Atomic force microscopy imaging and ion mobility mass spectrometry confirmed the formation of these latter midsize spherical particles at elevated pH. All these VP1 oligomers could be reversely assembled into the original capsid (VP1180). From the MS data collected over a range of experimental conditions, we suggest a disassembly model in which the T = 3 VP1180 particles dissociate into smaller oligomers, predominantly dimers, upon alkaline treatment prior to reassembly into VP160 and VP180 species.

Prestress Strengthens the Shell of Norwalk Virus Nanoparticles

We investigated the influence of the protruding domain of Norwalk virus-like particles (NVLP) on its overall structural and mechanical stability. Deletion of the protruding domain yields smooth mutant particles and our AFM nanoindentation measurements show a surprisingly altered indentation response of these particles. Notably, the brittle behavior of the NVLP as compared to the plastic behavior of the mutant reveals that the protruding domain drastically changes the capsids material properties. We conclude that the protruding domain introduces prestress, thereby increasing the stiffness of the NVLP and effectively stabilizing the viral nanoparticles. Our results exemplify the variety of methods that nature has explored to improve the mechanical properties of viral capsids, which in turn provides new insights for developing rationally designed, self-assembled nanodevices.

In vitro-reconstituted nucleoids can block mitochondrial DNA replication and transcription.

The mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000-10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A (TFAM). Here, we reconstitute nucleoid-like particles in vitro and demonstrate that small changes in TFAM levels dramatically impact the fraction of DNA molecules available for transcription and DNA replication. Compaction by TFAM is highly cooperative, and at physiological ratios of TFAM to DNA, there are large variations in compaction, from fully compacted nucleoids to naked DNA. In compacted nucleoids, TFAM forms stable protein filaments on DNA that block melting and prevent progression of the replication and transcription machineries. Based on our observations, we suggest that small variations in the TFAM-to-mtDNA ratio may be used to regulate mitochondrial gene transcription and DNA replication.

89Zr-Nanocolloidal Albumin–Based PET/CT Lymphoscintigraphy for Sentinel Node Detection in Head and Neck Cancer: Preclinical Results

Identifying sentinel nodes near the primary tumor remains a problem in, for example, head and neck cancer because of the limited resolution of current lymphoscintigraphic imaging when using 99mTc-nanocolloidal albumin. This study describes the development and evaluation of a nanocolloidal albumin–based tracer specifically dedicated for high-resolution PET detection. Methods: 89Zr was coupled to nanocolloidal albumin via the bifunctional chelate p-isothiocyanatobenzyldesferrioxamine B. Quality control tests, including particle size measurements, and in vivo biodistribution and imaging experiments in a rabbit lymphogenic metastasis model were performed. Results: Coupling of 89Zr to nanocolloidal albumin appeared to be efficient, resulting in a stable product with a radiochemical purity greater than 95%, without affecting the particle size. PET showed distinguished uptake of 89Zr-nanocolloidal albumin in the sentinel nodes, with visualization of lymphatic vessels, and with a biodistribution comparable to 99mTc-nanocolloidal albumin. Conclusion: 89Zr-nanocolloidal albumin is a promising tracer for sentinel node detection by PET.

Probing the impact of loading rate on the mechanical properties of viral nanoparticles

The effects of changes in the loading rate during the forced dissociation of single bonds have been studied for a wide variety of interactions. Less is known on the loading rate dependent behaviour of more complex systems that consist of multiple bonds. Here we focus on viral nanoparticles, in particular the protein shell (capsid) that protects the viral genome. As model systems we use the well-studied capsids of the plant virus Cowpea Chlorotic Mottle Virus (CCMV) and of the bacteriophages φ29 and HK97. By applying an atomic force microscopy (AFM) nanoindentation approach we study the loading rate dependency of their mechanical properties. Our AFM results show very diverse behaviour for the different systems. In particular, we find that not only the breaking force, but also the spring constant of some capsids depend on the loading rate. We describe and compare the measured data with simulation results from the literature. The unexpected complex loading rate dependencies that we report present a challenge for the current theoretical considerations aimed at understanding the molecular level interactions of highly ordered protein assemblies.

Sampling Protein Form and Function with the Atomic Force Microscope

To study the structure, function, and interactions of proteins, a plethora of techniques is available. Many techniques sample such parameters in non-physiological environments (e.g. in air, ice, or vacuum). Atomic force microscopy (AFM), however, is a powerful biophysical technique that can probe these parameters under physiological buffer conditions. With the atomic force microscope operating under such conditions, it is possible to obtain images of biological structures without requiring labeling and to follow dynamic processes in real time. Furthermore, by operating in force spectroscopy mode, it can probe intramolecular interactions and binding strengths. In structural biology, it has proven its ability to image proteins and protein conformational changes at submolecular resolution, and in proteomics, it is developing as a tool to map surface proteomes and to study protein function by force spectroscopy methods. The power of AFM to combine studies of protein form and protein function enables bridging various research fields to come to a comprehensive, molecular level picture of biological processes. We review the use of AFM imaging and force spectroscopy techniques and discuss the major advances of these experiments in further understanding form and function of proteins at the nanoscale in physiologically relevant environments.

FTIR Spectroscopy Revealing Light-Dependent Refolding of the Conserved Tongue Region of Bacteriophytochrome.

Bacteriophytochromes (BphPs) constitute a class of photosensory proteins that toggle between Pr and Pfr functional states through absorption of red and far-red light. The photosensory core of BphPs is composed of PAS, GAF, and PHY domains. Here, we apply FTIR spectroscopy to investigate changes in the secondary structure of Rhodopseudomonas palustris BphP2 (RpBphP2) upon Pr to Pfr photoconversion. Our results indicate conversion from a β-sheet to an α-helical element in the so-called tongue region of the PHY domain, consistent with recent X-ray structures of Deinococcus radiodurans DrBphP in dark and light states (TakalaH.; et al. Nature2014, 509, 245−24824776794). A conserved Asp in the GAF domain that noncovalently connects with the PHY domain and a conserved Pro in the tongue region of the PHY domain are essential for the β-sheet-to-α-helix conversion.

Mechanical Unfolding of an Autotransporter Passenger Protein Reveals the Secretion Starting Point and Processive Transport Intermediates

The backbone of secreted autotransporter passenger proteins generally attains a stable β-helical structure. The secretion of passengers across the outer membrane was proposed to be driven by sequential folding of this structure at the cell surface. This mechanism would require a relatively stable intermediate as starting point. Here, we investigated the mechanics of secreted truncated versions of the autotransporter hemoglobin protease (Hbp) of Escherichia coli using atomic force microscopy. The data obtained reveal a β-helical structure at the C terminus that is very stable. In addition, several other distinct metastable intermediates are found which are connected during unfolding by multiroute pathways. Computational analysis indicates that these intermediates correlate to the β-helical rungs in the Hbp structure which are clamped by stacked aromatic residues. Our results suggest a secretion mechanism that is initiated by a stable C-terminal structure and driven forward by several folding intermediates that build up the β-helical backbone.