We have studied the role of protein dynamics in chemical catalysis in the enzyme dihydrofolate reductase (DHFR), using a pump-probe method that employs pulsed-laser photothermal heating of a gold ...nanoparticle (AuNP) to directly excite a local region of the protein structure and transient absorbance to probe the effect on enzyme activity. Enzyme activity is accelerated by pulsed-laser excitation when the AuNP is attached close to a network of coupled motions in DHFR (on the FG loop, containing residues 116-132, or on a nearby alpha helix). No rate acceleration is observed when the AuNP is attached away from the network (distal mutant and His-tagged mutant) with pulsed excitation, or for any attachment site with continuous wave excitation. We interpret these results within an energy landscape model in which transient, site-specific addition of energy to the enzyme speeds up the search for reactive conformations by activating motions that facilitate this search.
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The catalytic reduction of CO2 is of great current interest because of its role in climate change and the energy cycle. We report a pterin electrocatalyst, 6,7-dimethyl-4-hydroxy-2-mercaptopteridine ...(PTE), that catalyzes the reduction of CO2 and formic acid on a glassy carbon electrode. Pterins are natural cofactors for a wide range of enzymes, functioning as redox mediators and C1 carriers, but they have not been exploited as electrocatalysts. Bulk electrolysis of a saturated CO2 solution in the presence of the PTE catalyst produces methanol, as confirmed by gas chromatography and 13C NMR spectroscopy, with a Faradaic efficiency of 10–23%. FTIR spectroelectrochemistry detected a progression of two-electron reduction products during bulk electrolysis, including formate, aqueous formaldehyde, and methanol. A transient intermediate was also detected by FTIR and tentatively assigned as a PTE carbamate. The results demonstrate that PTE catalyzes the reduction of CO2 at low overpotential and without the involvement of any metal.
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Importance of Membrane Fusion in Biology Membrane fusion is ubiquitous in biology, both in natural cellular functions such as neurotransmission and in pathological processes such as viral infection. ...The fusion of lipid bilayer membranes involves membrane contact, merger, and formation of an aqueous fusion pore, allowing the merging of separate compartments and mixing of their contents. For example, in neurotransmission, the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) mediates fusion of synaptic vesicles with the plasma membrane to release neurotransmitters, driven by zipping of the fourhelix bundle of the SNARE complex. Influenza hemagglutinin (HA) has served as a paradigm for understanding the mechanism of protein-mediated membrane fusion, and it is also an important target for antiviral drug development. HA is postulated to undergo an astounding series of refolding reactions triggered by lowered pH. The most remarkable aspect of the proposed mechanism is that it appears to be driven by HA refolding from a kinetically trapped, high-energy intermediate state
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Allosteric interactions in proteins generally involve propagation of local structural changes through the protein to a remote site. Anisotropic energy transport is thought to couple the remote sites, ...but the nature of this process is poorly understood. Here, we report the relationship between energy flow through the structure of bovine serum albumin and allosteric interactions between remote ligand binding sites of the protein. Ultrafast infrared spectroscopy is used to probe the flow of energy through the protein backbone following excitation of a heater dye, a metalloporphyrin or malachite green, bound to different binding sites in the protein. We observe ballistic and anisotropic energy flow through the protein structure following input of thermal energy into the flexible ligand binding sites, without local heating of the rigid helix bundles that connect these sites. This efficient energy transport mechanism enables the allosteric propagation of binding energy through the connecting helix structures.
Small single domain proteins that fold on the microsecond time scale have been the subject of intense interest as models for probing the complexity of folding energy landscapes. The villin headpiece ...subdomain (HP36) has been extensively studied because of its simple three helix structure, ultrafast folding lifetime of a few microseconds, and stable native fold. We have previously shown that folding as measured by a single 13C18O isotopic label on residue A57 in helix 2 occurs at a different rate than that measured by global probes of folding, indicating noncooperative complexity in the folding of HP36. In order to determine whether this complexity reflects intermediates or parallel pathways over a small activation barrier, 13C18O labels were individually incorporated at six different positions in HP36, including into all 3 helices. The equilibrium thermal unfolding transitions and the folding/unfolding dynamics were monitored using the unique IR signature of the 13C18O label by temperature dependent FTIR and temperature jump IR spectroscopy, respectively. Equilibrium experiments reveal that the 13C18O labels at different positions in HP36 show drastic differences in the midpoint of their transitions (T m), ranging from 45 to 67 °C. Heterogeneity is also observed in the relaxation kinetics; there are differences in the microsecond phase when different labeled positions are probed. At a final temperature of 45 °C, the relaxation rate for 13C18O A57 is 2.4e + 05 s–1 whereas for 13C18O L69 HP36 the relaxation rate is 5.1e + 05 s–1, two times faster. The observation of site-dependent midpoints for the equilibrium unfolding transitions and differences in the relaxation rates of the labeled positions enables us to probe the progressive accumulation of the folded structure, providing insight into the microscopic details of the folding mechanism.
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The fabrication of dynamic, transformable biomaterials that respond to environmental cues represents a significant step forward in the development of synthetic materials that rival their highly ...functional, natural counterparts. Here, we describe the design and synthesis of crystalline supramolecular architectures from charge-complementary heteromeric pairs of collagen-mimetic peptides (CMPs). Under appropriate conditions, CMP pairs spontaneously assemble into either 1D ultraporous (pore diameter >100 nm) tubes or 2D bilayer nanosheets due to the structural asymmetry that arises from heteromeric self-association. Crystalline collagen tubes represent a heretofore unobserved morphology of this common biomaterial. In-depth structural characterization from a suite of biophysical methods, including TEM, AFM, high-resolution cryo-EM, and SAXS/WAXS measurements, reveals that the sheet and tube assemblies possess a similar underlying lattice structure. The experimental evidence suggests that the tubular structures are a consequence of the self-scrolling of incipient 2D layers of collagen triple helices and that the scrolling direction determines the formation of two distinct structural isoforms. Furthermore, we show that nanosheets and tubes can spontaneously interconvert through manipulation of the assembly pH and systematic adjustment of the CMP sequence. Altogether, we establish initial guidelines for the construction of dynamically responsive 1D and 2D assemblies that undergo a structurally programmed morphological transition.
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A series of viologen related redox mediators of varying reduction potential has been characterized and their utility as electron shuttles between CdSe quantum dots and hydrogenase enzyme has been ...demonstrated. Tuning the mediator LUMO energy optimizes the performance of this hybrid photocatalytic system by balancing electron transfer rates of the shuttle.
The rate of the volume-phase transition for stimuli-responsive hydrogel particles ranging in size from millimeters to nanometers is limited by the rate of water transport, which is proportional to ...the surface area of the particle. Here, we hypothesized that the rate of volume-phase transition could be accelerated if the stimulus is geometrically controlled from the inside out, thus facilitating outward water ejection. To test this concept, we applied transient absorption spectroscopy, laser temperature-jump spectroscopy, and finite-element analysis modeling to characterize the dynamics of the volume-phase transition of hydrogel particles with a gold nanorod core. Our results demonstrate that the nanoscale heating of the hydrogel particle core led to an ultrafast, 60 ns particle collapse, which is 2–3 orders of magnitude faster than the response generated from conventional heating. This is the fastest recorded response time of a hydrogel material, thus opening potential applications for such stimuli-responsive materials.
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Many enzymes are known to change conformations during their catalytic cycle, but the role of these protein motions is not well understood.
dihydrofolate reductase (DHFR) is a small, flexible enzyme ...that is often used as a model system for understanding enzyme dynamics. Recently, native tryptophan fluorescence was used as a probe to study micro- to millisecond dynamics of DHFR. Yet, because DHFR has five native tryptophans, the origin of the observed conformational changes could not be assigned to a specific region within the enzyme. Here, we use DHFR mutants, each with a single tryptophan as a probe for temperature jump fluorescence spectroscopy, to further inform our understanding of DHFR dynamics. The equilibrium tryptophan fluorescence of the mutants shows that each tryptophan is in a different environment and that wild-type DHFR fluorescence is not a simple summation of all the individual tryptophan fluorescence signatures due to tryptophan-tryptophan interactions. Additionally, each mutant exhibits a two-phase relaxation profile corresponding to ligand association/dissociation convolved with associated conformational changes and a slow conformational change that is independent of ligand association and dissociation, similar to the wild-type enzyme. However, the relaxation rate of the slow phase depends on the location of the tryptophan within the enzyme, supporting the conclusion that the individual tryptophan fluorescence dynamics do not originate from a single collective motion, but instead report on local motions throughout the enzyme.
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The movement of protons and electrons is common to the synthesis of all chemical fuels such as H2. Hydrogenases, which catalyze the reversible reduction of protons, necessitate transport and ...reactivity between protons and electrons, but a detailed mechanism has thus far been elusive. Here, we use a phototriggered chemical potential jump method to rapidly initiate the proton reduction activity of a NiFe hydrogenase. Coupling the photochemical initiation approach to nanosecond transient infrared and visible absorbance spectroscopy afforded direct observation of interfacial electron transfer and active site chemistry. Tuning of intramolecular proton transport by pH and isotopic substitution revealed distinct concerted and stepwise proton-coupled electron transfer mechanisms in catalysis. The observed heterogeneity in the two sequential proton-associated reduction processes suggests a highly engineered protein environment modulating catalysis and implicates three new reaction intermediates; Nia-I, Nia-D, and Nia-SR–. The results establish an elementary mechanistic understanding of catalysis in a NiFe hydrogenase with implications in enzymatic proton-coupled electron transfer and biomimetic catalyst design.
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