Distinct Activation Mechanisms of CXCR4 and ACKR3 Revealed by Single-Molecule Analysis of their Conformational Landscapes.

The canonical chemokine receptor CXCR4 and atypical receptor ACKR3 both respond to CXCL12 but induce different effector responses to regulate cell migration. While CXCR4 couples to G proteins and directly promotes cell migration, ACKR3 is G protein-independent and scavenges CXCL12 to regulate extracellular chemokine levels and maintain CXCR4 responsiveness, thereby indirectly influencing migration. The receptors also have distinct activation requirements.

Early HIV-1 Gag Assembly on Lipid Membrane with vRNA.

HIV-1 capsid assembly is an essential process in the virus infection cycle. Initiation of capsid assembly involves viral proteins, genomic RNA, and the inner leaflet of the plasma membrane, facilitated by a number of cellular factors. The viral structural protein Gag plays a number of central roles in this process, including association with the membrane, selective binding of genomic RNA, and oligomerization and packaging to ultimately produce an immature budded pro-viral particle.

Early HIV-1 Gag Assembly on Lipid Membrane with vRNA.

Mass photometry (MP) was used to investigate the assembly of myristoylated full-length HIV-1 Gag (myr-Gag) and vRNA 5’ UTR fragment in a supported lipid bilayer (SLB) model system. The MP trajectories demonstrated that Gag trimerization on the membrane is a key step of early Gag assembly in the presence of vRNA. Growth of myr-Gag oligomers requires vRNA, occuring by addition of 1 or 2 monomers at a time from solution.

Conformational Dynamics of DNA Polymerases Revealed at the Single-Molecule Level.

DNA polymerases are intrinsically dynamic macromolecular machines. The purpose of this review is to describe the single-molecule Förster resonance energy transfer (smFRET) methods that are used to probe the conformational dynamics of DNA polymerases, focusing on DNA polymerase I. The studies reviewed here reveal the conformational dynamics underpinning the nucleotide selection, proofreading and 5' nuclease activities of Pol I.

Single-molecule view of coordination in a multi-functional DNA polymerase.

Replication and repair of genomic DNA requires the actions of multiple enzymatic functions that must be coordinated in order to ensure efficient and accurate product formation. Here, we have used single-molecule FRET microscopy to investigate the physical basis of functional coordination in DNA polymerase I (Pol I) from , a key enzyme involved in lagging-strand replication and base excision repair. Pol I contains active sites for template-directed DNA polymerization and 5' flap processing in separate domains.

Discrimination between Functional and Non-functional Cellular Gag Complexes involved in HIV-1 Assembly.

HIV-1 Gag and Gag-Pol are responsible for viral assembly and maturation and represent a major paradigm for enveloped virus assembly. Numerous intracellular Gag-containing complexes (GCCs) have been identified in cellular lysates using sucrose gradient ultracentrifugation. While these complexes are universally present in Gag-expressing cells, their roles in virus assembly are not well understood.

Biased Signaling of the G-Protein-Coupled Receptor βAR Is Governed by Conformational Exchange Kinetics.

G-protein-coupled receptors (GPCRs) mediate a wide range of human physiological functions by transducing extracellular ligand binding events into intracellular responses. GPCRs can activate parallel, independent signaling pathways mediated by G proteins or β-arrestins. Whereas "balanced" agonists activate both pathways equally, "biased" agonists dominantly activate one pathway, which is of interest for designing GPCR-targeting drugs because it may mitigate undesirable side effects.

Fluorophore Labeling, Nanodisc Reconstitution and Single-molecule Observation of a G Protein-coupled Receptor.

Activation of G protein-coupled receptors (GPCRs) by agonist ligands is mediated by a transition from an inactive to active receptor conformation. We describe a novel single-molecule assay that monitors activation-linked conformational transitions in individual GPCR molecules in real-time. The receptor is site-specifically labeled with a Cy3 fluorescence probe at the end of trans-membrane helix 6 and reconstituted in phospholipid nanodiscs tethered to a microscope slide.

A Survey of DDX21 Activity During Rev/RRE Complex Formation.

HIV-1 requires a specialized nuclear export pathway to transport unspliced and partially spliced viral transcripts to the cytoplasm. Central to this pathway is the viral protein Rev, which binds to the Rev response element in stem IIB located on unspliced viral transcripts and subsequently oligomerizes in a cooperative manner. Previous work identified a number of cellular DEAD-box helicases as in vivo binding partners of Rev, and siRNA experiments indicated a functional role for many in the HIV replication cycle.

A DEAD-box protein acts through RNA to promote HIV-1 Rev-RRE assembly.

The HIV-1 Rev protein activates nuclear export of unspliced and partially spliced viral RNA transcripts, which encode the viral genome and the genes encoding viral structural proteins, by binding to and oligomerizing on the Rev Response Element (RRE). The human DEAD-box protein 1 (DDX1) enhances the RNA export activity of Rev through an unknown mechanism. Using a single-molecule assembly assay and various DDX1 mutants, we show that DDX1 acts through the RRE RNA to specifically accelerate the nucleation step of the Rev-RRE assembly process.

A DEAD-Box Helicase Mediates an RNA Structural Transition in the HIV-1 Rev Response Element.

Nuclear export of partially spliced or unspliced HIV-1 RNA transcripts requires binding of the viral protein regulator of expression of virion (Rev) to the Rev response element (RRE) and subsequent oligomerization in a cooperative manner. Cellular DEAD-box helicase DEAD-box protein 1 (DDX1) plays a role in HIV replication, interacting with and affecting Rev-containing HIV transcripts in vivo, interacting directly with the RRE and Rev in vitro, and promoting Rev oligomerization in vitro. Binding of DDX1 results in enhancement of Rev oligomerization on the RRE that is correlated with an RNA structural change within the RRE that persists even after dissociation of DDX1.

FRET Characterization of Complex Conformational Changes in a Large 16S Ribosomal RNA Fragment Site-Specifically Labeled Using Unnatural Base Pairs.

Ribosome assembly has been studied intensively using Förster resonance energy transfer (FRET) with fluorophore-labeled fragments of RNA produced by chemical synthesis. However, these studies are limited by the size of the accessible RNA fragments. We have developed a replicable unnatural base pair (UBP) formed between (d)5SICS and (d)MMO2 or (d)NaM, which efficiently directs the transcription of RNA containing unnatural nucleotides.

Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor β2AR.

Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCR-targeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β2-adrenergic receptor (β2AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states.

Patient-reported perception of difficulty as a clinical indicator of dysfunctional neuromuscular control during the prone hip extension test and active straight leg raise test.

Two clinical tests used to assess for neuromuscular control deficits in patients with low back pain (LBP) are the prone hip extension (PHE) test and active straight leg raise (ASLR) test. For these tests, it has been suggested that patients be classified as "positive" if they demonstrate specific "abnormal" lumbopelvic motion patterns. For the ASLR test, the use of patient-reported perception of difficulty is also used to assess neuromuscular control.

Inter-rater agreement, sensitivity, and specificity of the prone hip extension test and active straight leg raise test.

Background: Two clinical tests used to assess for neuromuscular control deficits in low back pain (LBP) patients are the prone hip extension (PHE) test and active straight leg raise (ASLR) test. For these tests, it has been suggested examiners classify patients as "positive" or "negative" based on the presence or absence (respectively) of specific "abnormal" lumbopelvic motion patterns. The inter-rater agreement of such a classification scheme has been reported for the PHE test, but not for the ASLR test.

Vectorized data acquisition and fast triple-correlation integrals for Fluorescence Triple Correlation Spectroscopy.

Fluorescence Correlation Spectroscopy (FCS) is widely used to quantitate reaction rates and concentrations of molecules and . We recently reported Fluorescence Triple Correlation Spectroscopy (F3CS), which correlates three signals together instead of two. F3CS can analyze the stoichiometries of complex mixtures and detect irreversible processes by identifying time-reversal asymmetries.

Dynamics of site switching in DNA polymerase.

DNA polymerases replicate DNA by catalyzing the template-directed polymerization of deoxynucleoside triphosphate (dNTP) substrates onto the 3' end of a growing DNA primer strand. Many DNA polymerases also possess a separate 3'-5' exonuclease activity that is used to remove misincorporated nucleotides from the nascent DNA (proofreading). The polymerase (pol) and exonuclease (exo) activities are spatially separated in different enzyme domains, indicating that a mechanism must exist to transfer the growing primer terminus from one site to the other.

Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy.

The self-assembly of bacterial 30S ribosomes involves a large number of RNA folding and RNA-protein binding steps. The sequence of steps determines the overall assembly mechanism and the structure of the mechanism has ramifications for the robustness of biogenesis and resilience against kinetic traps. Thermodynamic interdependencies of protein binding inferred from omission-reconstitution experiments are thought to preclude certain assembly pathways and thus enforce ordered assembly, but this concept is at odds with kinetic data suggesting a more parallel assembly landscape.

Formation of trans-activation competent HIV-1 Rev:RRE complexes requires the recruitment of multiple protein activation domains.

The HIV-1 Rev trans-activator is a nucleocytoplasmic shuttle protein that is essential for virus replication. Rev directly binds to unspliced and incompletely spliced viral RNA via the cis-acting Rev Response Element (RRE) sequence. Subsequently, Rev oligomerizes cooperatively and interacts with the cellular nuclear export receptor CRM1.

Single-molecule Förster resonance energy transfer reveals an innate fidelity checkpoint in DNA polymerase I.

Enzymatic reactions typically involve complex dynamics during substrate binding, conformational rearrangement, chemistry, and product release. The noncovalent steps provide kinetic checkpoints that contribute to the overall specificity of enzymatic reactions. DNA polymerases perform DNA replication with outstanding fidelity by actively rejecting noncognate nucleotide substrates early in the reaction pathway.

The spectroscopic basis of fluorescence triple correlation spectroscopy.

We have developed fluorescence triple correlation spectroscopy (F3CS) as an extension of the widely used fluorescence microscopy technique fluorescence correlation spectroscopy. F3CS correlates three signals at once and provides additional capabilities for the study of systems with complex stoichiometry, kinetic processes, and irreversible reactions. A general theory of F3CS was developed to describe the interplay of molecular dynamics and microscope optics, leading to an analytical function to predict experimental triple correlations of molecules that freely diffuse through the tight focus of the microscope.

Single-molecule studies reveal that DEAD box protein DDX1 promotes oligomerization of HIV-1 Rev on the Rev response element.

Oligomeric assembly of Rev on the Rev response element (RRE) is essential for the nuclear export of unspliced and singly spliced human immunodeficiency virus type 1 viral mRNA transcripts. Several host factors, including the human DEAD box protein DDX1, are also known to be required for efficient Rev function. In this study, spontaneous assembly and dissociation of individual Rev-RRE complexes in the presence or absence of DDX1 were observed in real time via single-molecule total internal reflection fluorescence microscopy.

Resonance energy-transfer studies of the conformational change on the adsorption of oligonucleotides to a silica interface.

Time-resolved evanescent wave-induced fluorescence studies have been carried out on a series of fluorescently labeled oligonucleotide sequences adsorbed to a silica surface from solution. The fluorescence decay profiles of a fluorescent energy donor group undergoing resonance energy transfer to a nonemissive energy-acceptor molecule have been analyzed in terms of a distribution of donor-acceptor distances to reveal the conformational changes that occur in these oligonucleotides upon adsorption. Evanescent wave-induced time-resolved Förster resonance energy-transfer (EW-TRFRET) measurements indicate that at a high electrolyte concentration, there is localized separation of the oligonucleotide strands, and the helical structure adopts an "unraveled" conformation as a result of adsorption.