10 research outputs found
SymmetryĂą Directed SelfĂą Assembly of a Tetrahedral Protein Cage Mediated by de NovoĂą Designed Coiled Coils
The organization of proteins into new hierarchical forms is an important challenge in synthetic biology. However, engineering new interactions between protein subunits is technically challenging and typically requires extensive redesign of proteinĂą protein interfaces. We have developed a conceptually simple approach, based on symmetry principles, that uses short coiledĂą coil domains to assemble proteins into higherĂą order structures. Here, we demonstrate the assembly of a trimeric enzyme into a wellĂą defined tetrahedral cage. This was achieved by genetically fusing a trimeric coiledĂą coil domain to its C terminus through a flexible polyglycine linker sequence. The linker length and coiledĂą coil strength were the only parameters that needed to be optimized to obtain a high yield of correctly assembled protein cages.Geometry lesson: A modular approach for assembling proteins into largeĂą scale geometric structures was developed in which coiledĂą coil domains acted as Ăą twist tiesĂą to facilitate assembly. The geometry of the cage was specified primarily by the rotational symmetries of the coiled coil and building block protein and was largely independent of protein structural details.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138862/1/cbic201700406_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138862/2/cbic201700406.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138862/3/cbic201700406-sup-0001-misc_information.pd
Chemical Probes and Engineered Constructs Reveal a Detailed Unfolding Mechanism for a Solvent-Free Multidomain Protein
Despite
the growing application of gas-phase measurements in structural
biology and drug discovery, the factors that govern protein stabilities
and structures in a solvent-free environment are still poorly understood.
Here, we examine the solvent-free unfolding pathway for a group of
homologous serum albumins. Utilizing a combination of chemical probes
and noncovalent reconstructions, we draw new specific conclusions
regarding the unfolding of albumins in the gas phase, as well as more
general inferences regarding the sensitivity of collision induced
unfolding to changes in protein primary and tertiary structure. Our
findings suggest that the general unfolding pathway of low charge
state albumin ions is largely unaffected by changes in primary structure;
however, the stabilities of intermediates along these pathways vary
widely as sequences diverge. Additionally, we find that human albumin
follows a domain associated unfolding pathway, and we are able to
assign each unfolded form observed in our gas-phase data set to the
disruption of specific domains within the protein. The totality of
our data informs the first detailed mechanism for multidomain protein
unfolding in the gas phase, and highlights key similarities and differences
from the known solution-phase pathway
Ion Mobility-Mass Spectrometry Reveals a Dipeptide That Acts as a Molecular Chaperone for Amyloid ÎČ
Previously, we discovered and structurally
characterized a complex between amyloid ÎČ 1â40 and the
neuropeptide leucine enkephalin. This work identified leucine enkephalin
as a potentially useful starting point for the discovery of peptide-related
biotherapeutics for Alzheimerâs disease. In order to better
understand such complexes that are formed <i>in vitro</i>, we describe here the analysis of a series of site-directed amino
acid substitution variants of both peptides, covering the leucine
enkephalin sequence in its entirety and a large number of selected
residues of amyloid ÎČ 1â40 (residues: D1, E3, F4, R5,
H6, Y10, E11, H13, H14, Q15, K16, E22, K28, and V40). Ion mobilityâmass
spectrometry measurements and molecular dynamics simulations reveal
that the hydrophobic C-terminus of leucine enkephalin (Phe-Leu, FL)
is crucial for the formation of peptide complexes. As such, we explore
here the interaction of the dipeptide FL with both wildtype and variant
forms of amyloid ÎČ in order to structurally characterize the
complexes formed. We find that FL binds preferentially to amyloid
ÎČ oligomers and attaches to amyloid ÎČ within the region
between its N-terminus and its hydrophobic core, most specifically
at residues Y10 and Q15. We further show that FL is able to prevent
fibril formation
Evidence for a 1,3-Dipolar Cyclo-addition Mechanism in the Decarboxylation of Phenylacrylic Acids Catalyzed by Ferulic Acid Decarboxylase
Ferulic acid decarboxylase catalyzes
the decarboxylation of phenylacrylic
acid using a newly identified cofactor, prenylated flavin mononucleotide
(prFMN). The proposed mechanism involves the formation of a putative
pentacyclic intermediate formed by a 1,3 dipolar cyclo-addition of
prFMN with the αâÎČ double bond of the substrate,
which serves to activate the substrate toward decarboxylation. However,
enzyme-catalyzed 1,3 dipolar cyclo-additions are unprecedented and
other mechanisms are plausible. Here we describe the use of a mechanism-based
inhibitor, 2-fluoro-2-nitrovinylbenzene, to trap the putative cyclo-addition
intermediate, thereby demonstrating that prFMN can function as a dipole
in a 1,3 dipolar cyclo-addition reaction as the initial step in a
novel type of enzymatic reaction
Front Cover: SymmetryĂą Directed SelfĂą Assembly of a Tetrahedral Protein Cage Mediated by de NovoĂą Designed Coiled Coils (ChemBioChem 19/2017)
Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138873/1/cbic201700481.pd
Affinity-Based Selectivity Profiling of an In-Class Selective Competitive Inhibitor of Acyl Protein Thioesterase 2
Activity-based
protein profiling (ABPP) has revolutionized the
discovery and optimization of active-site ligands across distinct
enzyme families, providing a robust platform for in-class selectivity
profiling. Nonetheless, this approach is less straightforward for
profiling reversible inhibitors and does not access proteins outside
the ABPP probeâs target profile. While the active-site competitive
acyl protein thioesterase 2 inhibitor ML349 (<i>K</i><sub>i</sub> = 120 nM) is highly selective within the serine hydrolase
enzyme family, it could still interact with other cellular targets.
Here we present a chemoproteomic workflow to enrich and profile candidate
ML349-binding proteins. In human cell lysates, biotinylated-ML349
enriches a recurring set of proteins, including metabolite kinases
and flavin-dependent oxidoreductases that are potentially enhanced
by avidity-driven multimeric interactions. Confirmatory assays by
native mass spectrometry and fluorescence polarization quickly rank-ordered
these weak off-targets, providing justification to explore ligand
interactions and stoichiometry beyond ABPP
Freeze-out radii extracted from three-pion cumulants in pp, pâPb and PbâPb collisions at the LHC
In high-energy collisions, the spatio-temporal size of the particle production region can be measured using the Bose-Einstein correlations of identical bosons at low relative momentum. The source radii are typically extracted using two-pion correlations, and characterize the system at the last stage of interaction, called kinetic freeze-out. In low-multiplicity collisions, unlike in high-multiplicity collisions, two-pion correlations are substantially altered by background correlations, e.g. mini-jets. Such correlations can be suppressed using three-pion cumulant correlations. We present the first measurements of the size of the system at freeze-out extracted from three-pion cumulant correlations in pp, p-Pb and Pb-Pb collisions at the LHC with ALICE. At similar multiplicity, the invariant radii extracted in p-Pb collisions are found to be 5-15% larger than those in pp, while those in Pb-Pb are 35-55% larger than those in p-Pb. Our measurements disfavor models which incorporate substantially stronger collective expansion in p-Pb as compared to pp collisions at similar multiplicity
Production of inclusive Ï(1S) and Ï(2S) in pâPb collisions at âsNN = 5.02 TeV
We report on the production of inclusive ΄(1S) and ΄(2S) in p-Pb collisions at sNNââââ=5.02 TeV at the LHC. The measurement is performed with the ALICE detector at backward (â4.46<ycms<â2.96) and forward (2.03<ycms<3.53) rapidity down to zero transverse momentum. The production cross sections of the ΄(1S) and ΄(2S) are presented, as well as the nuclear modification factor and the ratio of the forward to backward yields of ΄(1S). A suppression of the inclusive ΄(1S) yield in p-Pb collisions with respect to the yield from pp collisions scaled by the number of binary nucleon-nucleon collisions is observed at forward rapidity but not at backward rapidity. The results are compared to theoretical model calculations including nuclear shadowing or partonic energy loss effects
Beauty production in pp collisions at âs = 2.76 TeV measured via semi-electronic decays
The ALICE collaboration at the LHC reports measurement of the inclusive production cross section of electrons from semi-leptonic decays of beauty hadrons with rapidity |y|<0.8 and transverse momentum 1<pT<10 GeV/c, in pp collisions at sâ= 2.76 TeV. Electrons not originating from semi-electronic decay of beauty hadrons are suppressed using the impact parameter of the corresponding tracks. The production cross section of beauty decay electrons is compared to the result obtained with an alternative method which uses the distribution of the azimuthal angle between heavy-flavour decay electrons and charged hadrons. Perturbative QCD calculations agree with the measured cross section within the experimental and theoretical uncertainties. The integrated visible cross section, Ïbâe=3.47±0.40(stat)+1.12â1.33(sys)±0.07(norm)ÎŒb, was extrapolated to full phase space using Fixed Order plus Next-to-Leading Log (FONLL) predictions to obtain the total bbÂŻ production cross section, ÏbbÂŻ=130±15.1(stat)+42.1â49.8(sys)+3.4â3.1(extr)±2.5(norm)±4.4(BR)ÎŒb