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 (MG) state can aid 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 MG 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 MG 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 MG 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 MG versus the fully ordered state. Additionally, these spectra represent the first demonstration of conserved helical content in a MG 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. Copyright © 2013 John Wiley & Sons, Ltd.     

Nativelike structure in designed four alpha-helix bundles driven by buried polar interactions

We show that a single internal polar interaction per helix is sufficient to engender structural specificity in that helix in helical bundle proteins. Furthermore, we use histidine-binding cofactors of different shapes which bind directly into the core, demonstrating that this structural specificity is not the result of a prescribed complimentary, "knobs in holes" core packing. We show that we can switch structural specificity of individual helices on and off by ligating cofactors, singly and in pairs, which bind either one or two histidine ligands. To our knowledge, this is the first demonstration of such extensive manipulation of protein structure by ligand binding, an important result of general interest to those working with self-assembled molecular systems. Finally, as these proteins were designed without the use of computational modeling, we not only demonstrate that designing a uniquely structured cofactor binding protein is not as difficult as is generally believed, we have determined why this is so: hydrophobic core complementarity, which is very difficult to design, is not necessary. Instead, a much simpler design process entails the creation of core polar interactions which themselves can drive conformational specificity.     

Chaotic Expansion of Elements of the Universal Enveloping Algebra of a Lie Algebra Associated with a Quantum Stochastic Calculus

The functional Ito formula, in the form df() = f( + d ) –f(),is formulated and proved in the context of a Lie algebra L associatedwith a quantum (non-commutative) stochastic calculus. Here fis an element of the universal enveloping algebra U of L, andf() + d() – f() is given a meaning using the coproductstructure of U even though the individual terms of this expressionhave no meaning. The Ito formula is equivalent to a chaoticexpansion formula for f() which is found explicitly. 1991 MathematicsSubject Classification: primary 81S25; secondary 60H05; tertiary18B25.     

Computational design of thermostabilizing D-amino acid substitutions

Judicious incorporation of D-amino acids in engineered proteins confers many advantages such as preventing degradation by endogenous proteases and promoting novel structures and functions not accessible to homochiral polypeptides. Glycine to D-alanine substitutions at the carboxy termini can stabilize α-helices by reducing conformational entropy. Beyond alanine, we propose additional side chain effects on the degree of stabilization conferred by D-amino acid substitutions. A detailed, molecular understanding of backbone and side chain interactions is important for developing rational, broadly applicable strategies in using D-amino acids to increase protein thermostability. Insight from structural bioinformatics combined with computational protein design can successfully guide the selection of stabilizing D-amino acid mutations. Substituting a key glycine in the Trp-cage miniprotein with D-Gln dramatically stabilizes the fold without altering the protein backbone. Stabilities of individual substitutions can be understood in terms of the balance of intramolecular forces both at the α-helix C-terminus and throughout the protein.     

Hydrogen bond-free flavin redox properties: managing flavins in extreme aprotic solvents

We report a simple, single step reaction that transforms riboflavin, which is insoluble in benzene, to tetraphenylacetyl riboflavin (TPARF), which is soluble in this solvent to over 250 mM. Electrochemical analysis of TPARF both alone and in complexes with two benzene-soluble flavin receptors: dibenzylamidopyridine (DBAP) and monobenzylamidopyridine (MBAP), prove that this model system behaves similarly to other previously studied flavin model systems which were soluble only in more polar solvents such as dichloromethane. Binding titrations in both benzene and dichloromethane show that the association constants of TPARF with its ligands are over an order of magnitude higher in benzene than dichloromethane, a consequence of the fact that benzene does not compete for H-bonds, but dichloromethane does. The alteration induced in the visible spectrum of TPARF in benzene upon the addition of DBAP is very similar to the difference produced by transfer to dichloromethane, further indicating that the flavin head group engages in a similar degree of hydrogen bonding with dichloromethane as with its ligands. This work underlines the importance of using a truly nonpolar solvent for the analysis of the effects of hydrogen bonding on the energetics of any biomimetic host-guest model system.     

15N solid-state NMR provides a sensitive probe of oxidized flavin reactive sites

Flavins are central to the reactivity of a wide variety of enzymes and electron transport proteins. There is great interest in understanding the basis for the different reactivities displayed by flavins in different protein contexts. We propose solid-state nuclear magnetic resonance (SS-NMR) as a tool for directly observing reactive positions of the flavin ring and thereby obtaining information on their frontier orbitals. We now report the SS-NMR signals of the redox-active nitrogens N1 and N5, as well as that of N3. The chemical shift tensor of N5 is over 720 ppm wide, in accordance with the predictions of theory and our calculations. The signal of N3 can be distinguished on the basis of coupling to 1H absent for N1 and N5, as well as the shift tensor span of only 170 ppm, consistent with N3's lower aromaticity and lack of a nonbonding lone pair. The isotropic shifts and spans of N5 and N1 reflect two opposite extremes of the chemical shift range for "pyridine-type" N's, consistent with their electrophilic and nucleophilic chemical reactivities, respectively. Upon flavin reduction, N5's chemical shift tensor contracts dramatically to a span of less than 110 ppm, and the isotropic chemical shift changes by approximately 300 ppm. Both are consistent with loss of N5's nonbonding lone pair and decreased aromaticity, and illustrate the responsiveness of the 15N chemical shift principal values to electronic structure. Thus. 15N chemical shift principal values promise to be valuable tools for understanding electronic differences that underlie variations in flavin reactivity, as well as the reactivities of other heterocyclic cofactors.