David S. Cafiso

Identifying conformational changes with site-directed spin labeling

Site-direct spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for detecting structural changes in proteins. This review provides examples that illustrate strategies for interpreting the data in terms of specific rearrangements in secondary and tertiary structure. The changes in the mobility and solvent accessibility of the spin label side chains, and in the distances between spin labels, report (i) rigid body motions of α-helices and β-strands (ii) relative movements of domains and (iii) changes in secondary structure. Such events can be monitored in the millisecond timescale, making it possible to follow structural changes during function. There is no upper limit to the size of proteins that can be investigated, and only 50–100 picomoles of protein are required. These features make site-directed spin labeling an attractive approach for the study of structure and dynamics in a wide range of systems.

Membrane structure and fusion-triggering conformational change of the fusion domain from influenza hemagglutinin.

The N-terminal domain of the influenza hemagglutinin (HA) is the only portion of the molecule that inserts deeply into membranes of infected cells to mediate the viral and the host cell membrane fusion. This domain constitutes an autonomous folding unit in the membrane, causes hemolysis of red blood cells and catalyzes lipid exchange between juxtaposed membranes in a pH-dependent manner. Combining NMR structures determined at pHs 7.4 and 5 with EPR distance constraints, we have deduced the structures of the N-terminal domain of HA in the lipid bilayer. At both pHs, the domain is a kinked, predominantly helical amphipathic structure. At the fusogenic pH 5, however, the domain has a sharper bend, an additional 310-helix and a twist, resulting in the repositioning of Glu 15 and Asp 19 relative to that at the nonfusogenic pH 7.4. Rotation of these charged residues out of the membrane plane creates a hydrophobic pocket that allows a deeper insertion of the fusion domain into the core of the lipid bilayer. Such an insertion mode could perturb lipid packing and facilitate lipid mixing between juxtaposed membranes.

Electrostatic Sequestration of PIP2 on Phospholipid Membranes by Basic/Aromatic Regions of Proteins

The basic effector domain of myristoylated alanine-rich C kinase substrate (MARCKS), a major protein kinase C substrate, binds electrostatically to acidic lipids on the inner leaflet of the plasma membrane; interaction with Ca2+/calmodulin or protein kinase C phosphorylation reverses this binding. Our working hypothesis is that the effector domain of MARCKS reversibly sequesters a significant fraction of the L-alpha-phosphatidyl-D-myo-inositol 4,5-bisphosphate (PIP2) on the plasma membrane. To test this, we utilize three techniques that measure the ability of a peptide corresponding to its effector domain, MARCKS(151-175), to sequester PIP2 in model membranes containing physiologically relevant fractions (15-30%) of the monovalent acidic lipid phosphatidylserine. First, we measure fluorescence resonance energy transfer from Bodipy-TMR-PIP2 to Texas Red MARCKS(151-175) adsorbed to large unilamellar vesicles. Second, we detect quenching of Bodipy-TMR-PIP2 in large unilamellar vesicles when unlabeled MARCKS(151-175) binds to vesicles. Third, we identify line broadening in the electron paramagnetic resonance spectra of spin-labeled PIP2 as unlabeled MARCKS(151-175) adsorbs to vesicles. Theoretical calculations (applying the Poisson-Boltzmann relation to atomic models of the peptide and bilayer) and experimental results (fluorescence resonance energy transfer and quenching at different salt concentrations) suggest that nonspecific electrostatic interactions produce this sequestration. Finally, we show that the PLC-delta1-catalyzed hydrolysis of PIP2, but not binding of its PH domain to PIP2, decreases markedly as MARCKS(151-175) sequesters most of the PIP2.

Internal electrostatic potentials in bilayers: measuring and controlling dipole potentials in lipid vesicles

The binding and translocation rates of hydrophobic cation and anion spin labels were measured in unilamellar vesicle systems formed from phosphatidylcholine. As a result of the membrane dipole potential, the binding and translocation rates for oppositely charged hydrophobic ions are dramatically different. These differences were analyzed using a simple electrostatic model and are consistent with the presence of a dipole potential of approximately 280 mV in phosphatidylcholine. Phloretin, a molecule that reduces the magnitude of the dipole potential, increases the translocation rate of hydrophobic cations, while decreasing the rate for anions. In addition, phloretin decreases the free energy of binding of the cation, while increasing the free energy of binding for the anion. The incorporation of 6-ketocholestanol also produces differential changes in the binding and translocation rates of hydrophobic ions, but in an opposite direction to those produced by phloretin. This is consistent with the view that 6-ketocholestanol increases the magnitude of the membrane dipole potential. A quantitative analysis of the binding and translocation rate changes produced by ketocholestanol and phloretin is well accounted for by a point dipole model that includes a dipole layer due to phloretin or 6-ketocholestanol in the membrane-solution interface. This approach allows dipole potentials to be estimated in membrane vesicle systems and permits predictable, quantitative changes in the magnitude of the internal electrostatic field in membranes. Using phloretin and 6-ketocholestanol, the dipole potential can be altered by over 200 mV in phosphatidylcholine vesicles.

NMR Solution Structure of the Integral Membrane Enzyme DsbB: Functional Insights into DsbB-Catalyzed Disulfide Bond Formation

We describe the NMR structure of DsbB, a polytopic helical membrane protein. DsbB, a bacterial cytoplasmic membrane protein, plays a key role in disulfide bond formation. It reoxidizes DsbA, the periplasmic protein disulfide oxidant, using the oxidizing power of membrane-embedded quinones. We determined the structure of an interloop disulfide bond form of DsbB, an intermediate in catalysis. Analysis of the structure and interactions with substrates DsbA and quinone reveals functionally relevant changes induced by these substrates. Analysis of the structure, dynamics measurements, and NMR chemical shifts around the interloop disulfide bond suggest how electron movement from DsbA to quinone through DsbB is regulated and facilitated. Our results demonstrate the extraordinary utility of NMR for functional characterization of polytopic integral membrane proteins and provide insights into the mechanism of DsbB catalysis.

Substrate-induced exposure of an energy-coupling motif of a membrane transporter.

BtuB is an outer membrane protein responsible for the uptake of vitamin B12 by Escherichia coli. It belongs to a family of bacterial transport proteins that derive energy for transport by coupling to the trans-periplasmic energy-coupling protein TonB. Using site-directed spin labeling and EPR we investigated the structure and substrate-induced changes in the TonB box, a highly conserved region in all TonB dependent transporters that may couple to TonB. In the absence of substrate, the line widths and collision parameters from EPR are consistent with this domain existing in a structured helical conformation that contacts the barrel of the transporter. Addition of substrate converts this segment into an extended structure that is highly dynamic, disordered and probably extended into the periplasm. This structural change demonstrates that the TonB box cycles between sequestered and accessible states in a substrate-dependent fashion. In a transport defective mutant of BtuB, this conformational cycle is disrupted and the TonB box appears to be extended even in the absence of substrate. These data suggest that the TonB box extends into the periplasm and interacts with TonB only in the presence of substrate.

Membrane orientation of the N-terminal segment of alamethicin determined by solid-state 15N NMR

Alamethicin was synthesized with 15N incorporated into alanine at position 6 in the peptide sequence. In dispersions of hydrated dimyristoylphosphatidylcholine, solid-state 15N NMR yields an axially symmetric powder pattern indicating that the peptide is reorienting with a single axis of symmetry when associated with lamellar lipids. When incorporated into bilayers that are uniformly oriented with the bilayer normal parallel to the B(o) field, the position of the observed 15N chemical shift is 171 ppm. This is coincident with the sigma parallel to edge of the axially symmetric powder pattern for non-oriented hydrated samples. Thus the axis of motional averaging lies along the bilayer normal. Two-dimensional separated local field spectra were obtained that provide a measure of the N-H dipolar coupling in one dimension and the 15N chemical shift in the other. These data yield a dipolar coupling of 17 kHz corresponding to an average angle of 24 degrees for the N-H bond with respect to the B(o) field axis. An analysis of the possible structures and orientations that could produce the observed spectral parameters show that these values are consistent with an alpha-helical conformation inserted along the bilayer normal.

Dynamic structure of lipid-bound synaptobrevin suggests a nucleation-propagation mechanism for trans-SNARE complex formation.

The synaptic vesicle protein synaptobrevin engages with syntaxin and SNAP-25 to form the SNARE complex, which drives membrane fusion in neuronal exocytosis. In the SNARE complex, the SNARE motif of synaptobrevin forms a 55-residue helix, but it has been assumed to be mostly unstructured in its prefusion form. NMR data for full-length synaptobrevin in dodecylphosphocholine micelles reveals two transient helical segments flanked by natively disordered regions and a third more stable helix. Transient helix I comprises the most N-terminal part of the SNARE motif, transient helix II extends the SNARE motif into the juxtamembrane region, and the more stable helix III is the transmembrane domain. These helices may have important consequences for SNARE complex folding and fusion: helix I likely forms a nucleation site, the C-terminal disordered SNARE motif may act as a folding arrest signal, and helix II likely couples SNARE complex folding and fusion.

Kinetics of Interaction of the Myristoylated Alanine-rich C Kinase Substrate, Membranes, and Calmodulin

Membrane binding of the myristoylated alanine-rich C kinase substrate (MARCKS) requires both its myristate chain and basic “effector” region. Previous studies with a peptide corresponding to the effector region, MARCKS-(151–175), showed that the 13 basic residues interact electrostatically with acidic lipids and that the 5 hydrophobic phenylalanine residues penetrate the polar head group region of the bilayer. Here we describe the kinetics of the membrane binding of fluorescent (acrylodan-labeled) peptides measured with a stopped-flow technique. Even though the peptide penetrates the polar head group region, the association of MARCKS-(151–175) with membranes is extremely rapid; association occurs with a diffusion-limited association rate constant. For example,k on = 1011 m −1 s−1 for the peptide binding to 100-nm diameter phospholipid vesicles. As expected theoretically,k on is independent of factors that affect the molar partition coefficient, such as the mole fraction of acidic lipid in the vesicle and the salt concentration. The dissociation rate constant (k off) is ∼10 s−1(lifetime = 0.1 s) for vesicles with 10% acidic lipid in 100 mm KCl. Ca2+-calmodulin (Ca2+·CaM) decreases markedly the lifetime of the peptide on vesicles, e.g. from 0.1 to 0.01 s in the presence of 5 μm Ca2+·CaM. Our results suggest that Ca2+·CaM collides with the membrane-bound MARCKS-(151–175) peptide and pulls the peptide off rapidly. We discuss the biological implications of this switch mechanism, speculating that an increase in the level of Ca2+-calmodulin could rapidly release phosphatidylinositol 4,5-bisphosphate that previous work has suggested is sequestered in lateral domains formed by MARCKS and MARCKS-(151–175).

Light-induced interfacial potentials in photoreceptor membranes

A rapid change in an interfacial electric potential of isolated bovine rod outer segment disk membranes occurs upon illumination. This potential change, which has been detected by the use of spin-labeled hydrophobic ions, apparently occurs within a low dielectric boundary region of the membrane near the external (cytoplasmic) surface and is positive with respect to the aqueous exterior of the disk. The magnitude of the potential change is pH-and temperature-dependent and appears with a first-order half-time of approximately 7 ms at 21 degrees C. A simple model in which one positive charge per bleached rhodopsin is translocated from the cytoplasmic aqueous space into the membrane low dielectric boundary region readily accounts for all experimental observations. The great similarity of the boundary potential change to the R2 phase of the early receptor potential suggests that the two have the same molecular origin.