Inbal Riven

Conformational Rearrangements Associated with the Gating of the G Protein-Coupled Potassium Channel Revealed by FRET Microscopy

G protein-coupled potassium channels (GIRK/Kir3.x) are key determinants that translate inhibitory chemical neurotransmission into changes in cellular excitability. To understand the mechanism of channel activation by G proteins, it is necessary to define the structural rearrangements in the channel that result from interaction with Gbetagamma subunits. In this study we used a combination of fluorescence spectroscopy and through-the-objective total internal reflection microscopy to monitor the conformational rearrangements associated with the activation of GIRK channels in single intact cells. We detect activation-induced changes in FRET consistent with a rotation and expansion of the termini along the central axis of the channel. We propose that this rotation and expansion of the termini drives the channel to open by bending and possibly rotating the second transmembrane segment.

Single-molecule fluorescence spectroscopy maps the folding landscape of a large protein

Proteins attain their function only after folding into a highly organized three-dimensional structure. Much remains to be learned about the mechanisms of folding of large multidomain proteins, which may populate metastable intermediate states on their energy landscapes. Here we introduce a novel method, based on high-throughput single-molecule fluorescence experiments, which is specifically geared towards tracing the dynamics of folding in the presence of a plethora of intermediates. We employ this method to characterize the folding reaction of a three-domain protein, adenylate kinase. Using thousands of single-molecule trajectories and hidden Markov modelling, we identify six metastable states on adenylate kinases folding landscape. Remarkably, the connectivity of the intermediates depends on denaturant concentration; at low concentration, multiple intersecting folding pathways co-exist. We anticipate that the methodology introduced here will find broad applicability in the study of folding of large proteins, and will provide a more realistic scenario of their conformational dynamics.

GIRK Channel Activation Involves a Local Rearrangement of a Preformed G Protein Channel Complex

G protein-coupled signaling is one of the major mechanisms for controlling cellular excitability. One of the main targets for this control at postsynaptic membranes is the G protein-coupled potassium channels (GIRK/Kir3), which generate slow inhibitory postsynaptic potentials following the activation of Pertussis toxin-sensitive G protein-coupled receptors. Using total internal reflection fluorescence (TIRF) microscopy combined with fluorescence resonance energy transfer (FRET), in intact cells, we provide evidence for the existence of a trimeric G protein-channel complex at rest. We show that activation of the channel via the receptor induces a local conformational switch of the G protein to induce channel opening. The presence of such a complex thus provides the means for a precise temporal and highly selective activation of the channel, which is required for fine tuning of neuronal excitability.

Three-dimensional localization of T-cell receptors in relation to microvilli using a combination of superresolution microscopies

Significance T lymphocytes play a central role in cell-mediated immunity. Their surfaces are covered by narrow and short protrusions called microvilli. It is not known whether there is a role for microvilli in the immune response. To shed light on this question, we probed the location of T-cell receptors (TCRs), the molecules that initiate the immune response of T cells, with respect to the 3D structure of microvilli. Superresolution optical microscopy showed that TCRs are highly concentrated on microvilli. Previous studies stressed the role of small clusters of TCRs in the immune process; our study provides a natural explanation as to how these clusters form. Leukocyte microvilli are flexible projections enriched with adhesion molecules. The role of these cellular projections in the ability of T cells to probe antigen-presenting cells has been elusive. In this study, we probe the spatial relation of microvilli and T-cell receptors (TCRs), the major molecules responsible for antigen recognition on the T-cell membrane. To this end, an effective and robust methodology for mapping membrane protein distribution in relation to the 3D surface structure of cells is introduced, based on two complementary superresolution microscopies. Strikingly, TCRs are found to be highly localized on microvilli, in both peripheral blood human T cells and differentiated effector T cells, and are barely found on the cell body. This is a decisive demonstration that different types of T cells universally localize their TCRs to microvilli, immediately pointing to these surface projections as effective sensors for antigenic moieties. This finding also suggests how previously reported membrane clusters might form, with microvilli serving as anchors for specific T-cell surface molecules.

Elucidation of the gating of the GIRK channel using a spectroscopic approach

The traditional view of G protein‐coupled receptor (GPCR)‐mediated signalling puts the players in this signalling cascade, namely the GPCR, the G protein and its effector, as individual components in space, where the signalling specificity is obtained mainly by the interaction of the GPCR and the Gα subunits of the G protein. A question is then raised as to how fidelity in receptor signalling is achieved, given that many systems use the same components of the G protein signalling machinery. One possible mechanism for obtaining the specific flow of the downstream signals, from the activated G protein to its specific effector target, in a timely manner, is compartmentalization, a spatial arrangement of the complex in a rather restricted space. Here we review our recent findings related to these issues, using the G protein‐coupled potassium channel (GIRK) as a model effector and fluorescence‐based approaches to reveal how the signalling complex is arranged and how the G protein exerts its action to activate the GIRK channel in intact cells.

Probing the Molecular Origin of Native-State Flexibility in Repeat Proteins.

In contrast to globular proteins, the structure of repeat proteins is dominated by a regular set of short-range interactions. This property may confer on the native state of such proteins significant elasticity. We probe the molecular origin of the spring-like behavior of repeat proteins using a designed tetratricopeptide repeat protein with three repeat units (CTPR3). Single-molecule fluorescence studies of variants of the protein with FRET pairs at different positions show a continuous expansion of the folded state of CTPR3 at low concentrations of a chemical denaturant, preceding the all-or-none transition to the unfolded state. This remarkable native-state expansion can be explained quantitatively by a reduction in the spring constant of the structure. Circular dichroism and tryptophan fluorescence spectroscopy further show that the expansion does not involve either unwinding of CTPR3 helices or unraveling of interactions within repeats. These findings point to hydrophobic inter-repeat contacts as the source of the elasticity of repeat proteins.

In vitro suppression of two different stop codons

Proteins play a crucial role in all living organisms, with the 20 natural amino acids as their building blocks. Unnatural amino acids are synthetic derivatives of these natural building blocks. These amino acids have unique chemical or physical properties as a result of their specific side chain residues. Their incorporation into proteins through ribosomal translation in response to one of the stop codons has opened a new way to manipulate and study proteins by enabling new functionalities, thus expending the genetic code. Different unnatural amino acids have different functionalities, hence, the ability to incorporate two different unnatural amino acids, in response to two different stop codons into one protein is a useful tool in protein manipulation. This ability has been achieved previously only in in vivo translational systems, however, with limited functionality. Herein, we report the incorporation of two different unnatural amino acids in response to two different stop codons into one protein, utilizing a cell‐free protein synthesis system. Biotechnol. Bioeng. 2017;114: 1065–1073.

Two states or not two states: Single-molecule folding studies of protein L

Experimental tools of increasing sophistication have been employed in recent years to study protein folding and misfolding. Folding is considered a complex process, and one way to address it is by studying small proteins, which seemingly possess a simple energy landscape with essentially only two stable states, either folded or unfolded. The B1-IgG binding domain of protein L (PL) is considered a model two-state folder, based on measurements using a wide range of experimental techniques. We applied single-molecule fluorescence resonance energy transfer (FRET) spectroscopy in conjunction with a hidden Markov model analysis to fully characterize the energy landscape of PL and to extract the kinetic properties of individual molecules of the protein. Surprisingly, our studies revealed the existence of a third state, hidden under the two-state behavior of PL due to its small population, ∼7%. We propose that this minority intermediate involves partial unfolding of the two C-terminal β strands of PL. Our work demonstrates that single-molecule FRET spectroscopy can be a powerful tool for a comprehensive description of the folding dynamics of proteins, capable of detecting and characterizing relatively rare metastable states that are difficult to observe in ensemble studies.

Direct observation of ultrafast large-scale dynamics of an enzyme under turnover conditions

Significance The potential effect of conformational dynamics of enzymes on their chemical steps has been intensely debated recently. We use single-molecule FRET experiments on adenylate kinase (AK) to shed new light on this question. AK closes its domains to bring its two substrate close together for reaction. We show that domain closure takes only microseconds to complete, which is two orders of magnitude faster than the chemical reaction. Nevertheless, active-site mutants that reduce the rate of domain closure also reduce the reaction rate, suggesting a connection between the two phenomena. We propose that ultrafast domain closure is used by enzymes as a mechanism to optimize mutual orientation of substrates, a novel mode of coupling between conformational dynamics and catalysis. The functional cycle of many proteins involves large-scale motions of domains and subunits. The relation between conformational dynamics and the chemical steps of enzymes remains under debate. Here we show that in the presence of substrates, domain motions of an enzyme can take place on the microsecond time scale, yet exert influence on the much-slower chemical step. We study the domain closure reaction of the enzyme adenylate kinase from Escherichia coli while in action (i.e., under turnover conditions), using single-molecule FRET spectroscopy. We find that substrate binding increases dramatically domain closing and opening times, making them as short as ∼15 and ∼45 µs, respectively. These large-scale conformational dynamics are likely the fastest measured to date, and are ∼100–200 times faster than the enzymatic turnover rate. Some active-site mutants are shown to fully or partially prevent the substrate-induced increase in domain closure times, while at the same time they also reduce enzymatic activity, establishing a clear connection between the two phenomena, despite their disparate time scales. Based on these surprising observations, we propose a paradigm for the mode of action of enzymes, in which numerous cycles of conformational rearrangement are required to find a mutual orientation of substrates that is optimal for the chemical reaction.

Manipulating the Folding Landscape of a Multidomain Protein

Folding of proteins to their functional conformation is paramount to life. Though 75% of the proteome consists of multidomain proteins, our knowledge of folding has been based primarily on studies conducted on single-domain and fast-folding proteins. Nonetheless, the complexity of folding landscapes exhibited by multidomain proteins has received increased scrutiny in recent years. We study the three-domain protein adenylate kinase from E. coli (AK), which has been shown to fold through a series of pathways involving several intermediate states. We use a protein design method to manipulate the folding landscape of AK, and single-molecule FRET spectroscopy to study the effects on the folding process. Mutations introduced in the NMP binding (NMPbind) domain of the protein are found to have unexpected effects on the folding landscape. Thus, while stabilizing mutations in the core of the NMPbind domain retain the main folding pathways of wild-type AK, a destabilizing mutation at the interface between the NMPbind and the CORE domains causes a significant repartition of the flux between the folding pathways. Our results demonstrate the outstanding plasticity of the folding landscape of AK and reveal how specific mutations in the primary structure are translated into changes in folding dynamics. The combination of methodologies introduced in this work should prove useful for deepening our understanding of the folding process of multidomain proteins.