41 research outputs found
Speeding up biomolecular interactions by molecular sledding
Numerous biological processes involve association of a protein with its binding partner, an event that is preceded by a diffusion-mediated search bringing the two partners together. Often hindered by crowding in biologically relevant environments, three-dimensional diffusion can be slow and result in long bimolecular association times. Similarly, the initial association step between two binding partners often represents a rate-limiting step in biotechnologically relevant reactions. We demonstrate the practical use of an 11-a.a. DNA-interacting peptide derived from adenovirus to reduce the dimensionality of diffusional search processes and speed up associations between biological macromolecules. We functionalize binding partners with the peptide and demonstrate that the ability of the peptide to one-dimensionally diffuse along DNA results in a 20-fold reduction in reaction time. We also show that modifying PCR primers with the peptide sled enables significant acceleration of standard PCR reactions
Structure and uncoating of immature adenovirus
Maturation via proteolytical processing is a common trait in the viral world, and is
often accompanied by large conformational changes and rearrangements in the capsid.
The adenovirus protease has been shown to play a dual role in the viral infectious
cycle: (a) in maturation, as viral assembly starts with precursors to several of the
structural proteins, but ends with proteolytically processed versions in the mature
virion; and (b) in entry, because protease-impaired viruses have difficulties in
endosome escape and uncoating. Indeed, viruses that have not undergone proteolytical
processing are not infectious. We present the 3D structure of immature adenovirus
particles, as represented by the thermosensitive mutant Ad2 ts1 grown under nonpermissive
conditions, and compare it with the mature capsid. Our 3DEM maps at
subnanometer resolution indicate that adenovirus maturation does not involve large
scale conformational changes in the capsid. Difference maps reveal the location of
unprocessed peptides pIIIa and pVI and help to define their role in capsid assembly
and maturation. An intriguing difference appears in the core, indicating a more
compact organization and increased stability of the immature cores. We have further
investigated these properties by in vitro disassembly assays. Fluorescence and
electron microscopy experiments reveal differences in the stability and uncoating of
immature viruses, both at the capsid and core levels, as well as disassembly
intermediates not previously imaged.This work was supported by grants from the Ministerio de Ciencia e Innovación of Spain (BFU2007-60228 to C.S.M. and BIO2007-67150-C03-03 to R.M.), the Comunidad Autónoma de Madrid and Consejo Superior de Investigaciones Científicas (CCG08-CSIC/SAL-3442 to C.S.M.) and the National Institutes of Health (5R01CA111569 to D.T.C., R0141599 to W.F.M. and GM037705 to S.J.F.). R.M.-C. is a recipient of a PFIS fellowship from the Instituto de Salud Carlos III of Spain. A.J.P.-B. holds a CSIC JAE-Doc postdoctoral position, partially funded by the European Social FundPeer reviewe
Structure, Function and Dynamics in Adenovirus Maturation
Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core is more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. Finally, possible roles for maturation of the terminal protein are discussed.This publication was supported
by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Awards Numbered R01AI41599 and R21AI113565, to Walter F. Mangel. Work at the Carmen San Martín laboratory was funded by grants BFU2010-16382, BFU2013-41249-P, and the Spanish Interdisciplinary Network on
the Biophysics of Viruses (Biofivinet, FIS2011-16090-E) from the Ministerio de Economía y
Competitividad of Spain.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)
Modulation of the Plasminogen Activator Activity of a Transformed Cell Line by Cell Density
Molecular sled is an eleven-amino acid vehicle facilitating biochemical interactions via sliding components along DNA
Recently, we showed the adenovirus proteinase interacts productively with its protein substrates in vitro and in vivo in nascent virus particles via one-dimensional diffusion along the viral DNA. The mechanism by which this occurs has heretofore been unknown. We show sliding of these proteins along DNA occurs on a new vehicle in molecular biology, a ‘molecular sled’ named pVIc. This 11-amino acid viral peptide binds to DNA independent of sequence. pVIc slides on DNA, exhibiting the fastest one-dimensional diffusion constant, 26±1.8 × 10[superscript 6] (bp)[superscript 2] s[superscript −1]. pVIc is a ‘molecular sled,’ because it can slide heterologous cargos along DNA, for example, a streptavidin tetramer. Similar peptides, for example, from the C terminus of β-actin or NLSIII of the p53 protein, slide along DNA. Characteristics of the ‘molecular sled’ in its milieu (virion, nucleus) have implications for how proteins in the nucleus of cells interact and imply a new form of biochemistry, one-dimensional biochemistry.Broad Institute of MIT and Harvard (Startup Funding)Burroughs Wellcome Fund (Career Award at the Scientific Interface
Nonisotopic DNA Detection System Employing Elastase and a Fluorogenic Rhodamine Substrate
An alternative fluorescence-based method has been developed for the direct detection of small quantities of DNA in solution. In this system, a serine protease (elastase) is coupled to a DNA oligonucleotide through a disulfide linkage. A bis(tetraalanine)-derivatized rhodamine molecule
(BZTAlaR) has been synthesized for use as a substrate. BZTAlaR is nonfluorescent in its derivatized form and shows negligible hydrolysis in solution. Cleavage of the tetraalanyl groups from the rhodamine portion of the molecule restores its fluorescence. Hybridization of the elastase-oligonucleotide conjugate to its target, capture of the conjugate-target complex with streptavidin-coated magnetic beads, addition of substrate, and subsequent detection of the target by fluorescence are accomplished in solution. Hybridization is rapid and specific, with over 90% of a target sequence successfully hybridized and captured. This method exhibits low background and an amplified fluorescent signal over time, resulting in a current detection limit of 0.49 fmol of elastase alone, or 2.64 fmol of conjugate, within 2 h
Nonisotopic DNA Detection System Employing Elastase and a Fluorogenic Rhodamine Substrate
An alternative fluorescence-based method has been developed for the direct detection of small quantities of DNA in solution. In this system, a serine protease (elastase) is coupled to a DNA oligonucleotide through a disulfide linkage. A bis(tetraalanine)-derivatized rhodamine molecule
(BZTAlaR) has been synthesized for use as a substrate. BZTAlaR is nonfluorescent in its derivatized form and shows negligible hydrolysis in solution. Cleavage of the tetraalanyl groups from the rhodamine portion of the molecule restores its fluorescence. Hybridization of the elastase-oligonucleotide conjugate to its target, capture of the conjugate-target complex with streptavidin-coated magnetic beads, addition of substrate, and subsequent detection of the target by fluorescence are accomplished in solution. Hybridization is rapid and specific, with over 90% of a target sequence successfully hybridized and captured. This method exhibits low background and an amplified fluorescent signal over time, resulting in a current detection limit of 0.49 fmol of elastase alone, or 2.64 fmol of conjugate, within 2 h