Biology subjects

Koder, R. L.

Publications and source records attributed to Koder, R. L..

4 recordsLinked to original sources

bioRxiv2023

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 which 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 which 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. SignificanceThis communication sheds light on one of the more controversial areas in protein folding and design: the dynamic nature of the hydrophobic core and its relationship to metalloprotein function, in particular the relationship between dynamic solvent penetration into the protein core and the stability of metalloenzyme intermediates. We demonstrate that the basic tetrameric scaffold that is the classic helical bundle model for cofactor binding and activation can be easily upgraded to a more rigid, less dynamic, single chain helical bundle by merely taking the same helical sequences and converting it to a single chain protein connected by simple, nonoptimized glycine-rich loops. Importantly, our results explain the decades-long history of failure in the design of proteins capable of stably forming an oxyferrous state - the requirement for a protein large enough to protect the heme porphyrin surface with both structural specificity and sufficient structural rigidity to restrict water penetration into the protein core. Finally, we believe this is the first use of Deep UV Resonance Raman spectroscopy to monitor dynamic water penetration in a functional protein. This method may prove useful moving forward to many research groups.

biophysics↗

bioRxiv2023

Elastin Recoil is Driven by the Hydrophobic Effect

Elastin is an extracellular matrix material found in all vertebrates. Its reversible elasticity, robustness and low stiffness are essential for the function of arteries, lungs, and skin. It is among the most resilient elastic materials known: During a human lifetime, arterial elastin undergoes in excess of 2x109 stretching/contracting cycles without replacement and slow oxidative hardening has been identified as a limiting factor on human lifespan. For over fifty years, the mechanism of entropic recoil has been controversial. Herein, we report a combined NMR and thermomechanical study that establishes the hydrophobic effect as the primary driver of elastin function. Water ordering at the solvent:protein interface was observed as a function of stretch using double quantum 2H NMR and the most extensive thermodynamic analysis performed to date was obtained by measuring elastin length and volume as a function of force and temperature in normal water, heavy water and with co-solvents. When stretched, elastins heat capacity increases, water is ordered proportional to the degree of stretching, the internal energy decreases, and heat is released in excess of the work performed. These properties show that recoil in elastin under physiological conditions is primarily driven by the hydrophobic effect rather than by configurational entropy as is the case for rubber. Consistent with this conclusion are decreases in the thermodynamic signatures when co-solvents that alter the hydrophobic effect are introduced. We propose that hydrophobic effect-driven recoil, as opposed to a configurational entropy mechanism, where hardening from crystallization can occur, is the origin of elastins unusual resilience. SignificanceElastin, found in tissues that require reversible elasticity, has low stiffness and great resiliency. It is a self-assembled material that has been a target for regenerative medicine. However, the basis for its elasticity has been controversial for more than 50 years. Formed from a hydrophobic protein with an equivalent mass of water, the controversy is whether recoil is driven by entropy gain of the protein and/or the water. We demonstrate that matrix water is progressively ordered upon stretching and that the thermodynamics of elastin recoil are those of the hydrophobic effect and different from those of rubber. We conclude that recoil is primarily driven by the hydrophobic effect and suggest that this accounts for elastins low stiffness and high resilience.

biophysics↗

bioRxiv2022

Design of a Minimal di-Nickel Hydrogenase Peptide

The most ancient processes for energy production in the evolution of life involve the reversible oxidation of molecular hydrogen by hydrogenase. Extant hydrogenase enzymes are complex, comprising hundreds of amino acids and multiple cofactors. We designed a 13 amino acid nickel-binding peptide capable of robustly producing molecular hydrogen from protons under a wide variety of conditions. The peptide forms a di-nickel cluster structurally analogous to a Ni-Fe cluster in [NiFe]-hydrogenase and the Ni-Ni cluster in acetyl-CoA synthase (ACS), two ancient, extant proteins central to metabolism. These experimental results clearly demonstrate that modern enzymes, despite their enormous complexity, likely evolved from simple peptide precursors on early Earth. One Sentence SummarySmall metal-binding peptides were the likely precursors of modern enzymes.

biochemistry↗

bioRxiv2022

Oxidation-reduction and photophysical properties of isomeric forms of Safranin

Safranine O is widely used in the bioenergetics community as an indicator dye to determine membrane potentials and as an electron transfer mediator in potentiometric titrations. Here we show that two different commercial preparations of Safranine O contain less than sixty percent by weight of the title compound, with the rest primarily consisting of two closely related safranine isomers. All three major isomer components were isolated using reverse phase HPLC and their structures determined using mass spectrometry and two-dimensional NMR. These Safranines have two-electron midpoint potentials ranging from -272 to -315 mV vs. SHE. We have also investigated the absorption and fluorescence spectra of the compounds and found that they display distinct spectral and photophysical properties. While this mixture may aid in Safranine Os utility as a mediator compound, membrane potential measurements must take this range of dye potentials into account.

biochemistry↗