Protein dynamics govern the oxyferrous state lifetime of an artificial oxygen transport protein.

It has long been known that the alteration of protein side chains that occlude or expose the heme cofactor to water can greatly affect the stability of the oxyferrous heme state. Here, we demonstrate that the rate of dynamically driven water penetration into the core of an artificial oxygen transport protein also correlates with oxyferrous state lifetime by reducing global dynamics, without altering the structure of the active site, via the simple linking of the two monomers in a homodimeric artificial oxygen transport protein using a glycine-rich loop. The tethering of these two helices does not significantly affect the active site structure, pentacoordinate heme-binding affinity, reduction potential, or gaseous ligand affinity. It does, however, significantly reduce the hydration of the protein core, as demonstrated by resonance Raman spectroscopy, backbone amide hydrogen exchange, and pKa shifts in buried histidine side chains. This further destabilizes the charge-buried entatic state and nearly triples the oxyferrous state lifetime. These data are the first direct evidence that dynamically driven water penetration is a rate-limiting step in the oxidation of these complexes. It furthermore demonstrates that structural rigidity that limits water penetration is a critical design feature in metalloenzyme construction and provides an explanation for both the failures and successes of earlier attempts to create oxygen-binding proteins.

Computational design of a sensitive, selective phase-changing sensor protein for the VX nerve agent.

The VX nerve agent is one of the deadliest chemical warfare agents. Specific, sensitive, real-time detection methods for this neurotoxin have not been reported. The creation of proteins that use biological recognition to fulfill these requirements using directed evolution or library screening methods has been hampered because its toxicity makes laboratory experimentation extraordinarily expensive. A pair of VX-binding proteins were designed using a supercharged scaffold that couples a large-scale phase change from unstructured to folded upon ligand binding, enabling fully internal binding sites that present the maximum surface area possible for high affinity and specificity in target recognition. Binding site residues were chosen using a new distributed evolutionary algorithm implementation in protCAD. Both designs detect VX at parts per billion concentrations with high specificity. Computational design of fully buried molecular recognition sites, in combination with supercharged phase-changing chassis proteins, enables the ready development of a new generation of small-molecule biosensors.

Unwinding of the Substrate Transmembrane Helix in Intramembrane Proteolysis.

Intramembrane-cleaving proteases (I-CLiPs) activate pools of single-pass helical membrane protein signaling precursors that are key in the physiology of prokaryotic and eukaryotic cells. Proteases typically cleave peptide bonds within extended or flexible regions of their substrates, and thus the mechanism underlying the ability of I-CLiPs to hydrolyze the presumably α-helical transmembrane domain (TMD) of these membrane proteins is unclear. Using deep-ultraviolet resonance Raman spectroscopy in combination with isotopic labeling, we show that although predominantly in canonical α-helical conformation, the TMD of the established I-CLiP substrate Gurken displays 310-helical geometry. As measured by microscale thermophoresis, this substrate binds with high affinity to the I-CLiPs GlpG rhomboid and MCMJR1 presenilin homolog in detergent micelles. Binding results in deep-ultraviolet resonance Raman spectra, indicating conformational changes consistent with unwinding of the 310-helical region of the substrate's TMD. This 310-helical conformation is key for intramembrane proteolysis, as the substitution of a single proline residue in the TMD of Gurken by alanine suppresses 310-helical content in favor of α-helical geometry and abolishes cleavage without affecting binding to the I-CLiP. Complemented by molecular dynamics simulations of the TMD of Gurken, our vibrational spectroscopy data provide biophysical evidence in support of a model in which the transmembrane region of cleavable I-CLiP substrates displays local deviations in canonical α-helical conformation characterized by chain flexibility, and binding to the enzyme results in conformational changes that facilitate local unwinding of the transmembrane helix for cleavage.

Bilayer surface association of the pHLIP peptide promotes extensive backbone desolvation and helically-constrained structures.

Despite their presence in many aspects of biology, the study of membrane proteins lags behind that of their soluble counterparts. Improving structural analysis of membrane proteins is essential. Deep-UV resonance Raman (DUVRR) spectroscopy is an emerging technique in this area and has demonstrated sensitivity to subtle structural transitions and changes in protein environment. The pH low insertion peptide (pHLIP) has three distinct structural states: disordered in an aqueous environment, partially folded and associated with a lipid membrane, and inserted into a lipid bilayer as a transmembrane helix. While the soluble and membrane-inserted forms are well characterized, the partially folded membrane-associated state has not yet been clearly described. The amide I mode, known to be sensitive to protein environment, is the same in spectra of membrane-associated and membrane-inserted pHLIP, indicating comparable levels of backbone dehydration. The amide S mode, sensitive to helical structure, indicates less helical character in the membrane-associated form compared to the membrane-inserted state, consistent with previous findings. However, the structurally sensitive amide III region is very similar in both membrane-associated and membrane-inserted pHLIP, suggesting that the membrane-associated form has a large amount of ordered structure. Where before the membrane-associated state was thought to contain mostly unordered structure and reside in a predominantly aqueous environment, we have shown that it contains a significant amount of ordered structure and rests deeper within the lipid membrane.

Observation of persistent α-helical content and discrete types of backbone disorder during a molten globule to ordered peptide transition via deep-UV resonance Raman spectroscopy.

The molten globule state can aide in the folding of a protein to a functional structure and is loosely defined as an increase in structural disorder with conservation of the ensemble secondary structure content. Simultaneous observation of persistent secondary structure content with increased disorder has remained experimentally problematic. As a consequence, modeling how the molten globule state remains stable and how it facilitates proper folding remains difficult due to a lack of amenable spectroscopic techniques to characterize this class of partially unfolded proteins. Previously, deep-UV resonance Raman (dUVRR) spectroscopy has proven useful in the resolution of global and local structural fluctuations in the secondary structure of proteins. In this work, dUVRR was employed to study the molten globule to ordered transition of a model four-helix bundle protein, HP7. Both the average ensemble secondary structure and types of local disorder were monitored, without perturbation of the solvent, pH, or temperature. The molten globule to ordered transition is induced by stepwise coordination of two heme molecules. Persistent dUVRR spectral features in the amide III region at 1295-1301 and 1335-1338 cm-1 confirm previous observations that HP7 remains predominantly helical in the molten globule versus the fully ordered state. Additionally, these spectra represent the first demonstration of conserved helical content in a molten globule protein. With successive heme binding significant losses are observed in the spectral intensity of the amide III3 and S regions (1230-1260 and 1390 cm-1, respectively), which are known to be sensitive to local disorder. These observations indicate that there is a decrease in the structural populations able to explore various extended conformations, with successive heme binding events. DUVRR spectra indicate that the first heme coordination between two helical segments diminishes exploration of more elongated backbone structural conformations in the inter-helical regions. A second heme coordination by the remaining two helices further restricts protein motion.