Protein-cofactor Interactions Research Articles

Tailorable Tetrahelical Bundles as a Toolkit for Redox Studies.

Oxidoreductases have evolved over millions of years to perform a variety of metabolic tasks crucial for life. Understanding how these tasks are engineered relies on delivering external electron donors or acceptors to initiate electron transfer reactions. This is a challenge. Small-molecule redox reagents can act indiscriminately, poisoning the cell. Natural redox proteins are more selective, but finding the right partner can be difficult due to the limited number of redox potentials and difficulty tuning them. De novo proteins offer an alternative path. They are robust and can withstand mutations that allow for tailorable changes. They are also devoid of evolutionary artifacts and readily bind redox cofactors. However, no reliable set of engineering principles have been developed that allow for these proteins to be fine-tuned so their redox midpoint potential (Em) can form donor/acceptor pairs with any natural oxidoreductase. This work dissects protein-cofactor interactions that can be tuned to modulate redox potentials of acceptors and donors using a mutable de novo designed tetrahelical protein platform with iron tetrapyrrole cofactors as a test case. We show a series of engineered heme b-binding de novo proteins and quantify their resulting effect on Em. By focusing on the surface charge and buried charges, as well as cofactor placement, chemical modification, and ligation of cofactors, we are able to achieve a broad range of Em values spanning a range of 330 mV. We anticipate this work will guide the design of proteinaceous tools that can interface with natural oxidoreductases inside and outside the cell while shedding light on how natural proteins modulate Em values of bound cofactors.

Systems analysis of steroid induced osteonecrosis shows role for heme and vitamin D in pathogenesis

ObjectiveFemoral Head Osteonecrosis (FHO) which includes Steroid-induced FHO (SFHO) is a debilitating disease. Though extensively studied the mechanisms have remained elusive. Hence systems analysis is carried out to identify a potential mechanism, biomarkers and therapeutic targets. DesignGene Set Enrichment Analysis (GSEA) and clustering analysis were performed on GEO data sets. A Cofactor-protein interaction network was generated and pathway analysis was performed. Followed by the generation of an integrated network model. Metabolic data were analyzed using MetaboAnalyst. ADMET analysis of steroids was carried out. ResultsSystems analyses based on GSEA, Pathway clustering and Cofactor gene network showed that the enriched pathways namely Steroid, Cholesterol, Vitamin D and heme metabolism could be regulated by Vitamin B6, B12, and heme cofactors. The integrated network model showed CYP450 genes which are critical for bioactivation of Vitamin D and clearing of xenobiotic steroid metabolites which are implicated in SFHO. Analysis of transcription factors and upstream kinome showed a role for HNF4A, SREBP and GSK3b in the pathogenesis. ADMET analysis identified CYP3A4 in SFHO associated steroid clearance. Vitamin D modulated genes CBS and CYP3A4 are downregulated in SFHO potentially due to deregulated vitamin D metabolism. GSEA analysis of steroid-treated osteoblasts and cartilage show enrichment of adipogenesis, cholesterol, xenobiotic metabolism, etc. ConclusionTaken together our analysis reveals the molecular signatures associated with the disease and identifies a critical role for vitamin D, B6, B12 and heme metabolism. Low B6, B12 modulate homocysteine levels and osteoclastogenesis, while compromised heme metabolism modulate CYP450s mediated steroid clearance, Vitamin D bioactivation and influence osteoblastogenesis. Thus steroids orchestrate the events that might influence disease pathogenesis by modulating bone remodeling and thrombosis. Furthermore, our work has implications for the protection of COVID-19 patients from osteonecrosis who underwent steroid therapy.

Thyroid hormone receptor β sumoylation is required for thyrotropin regulation and thyroid hormone production.

Thyroid hormone receptor β (THRB) is posttranslationally modified by small ubiquitin-like modifier (SUMO). We generated a mouse model with a mutation that disrupted sumoylation at lysine 146 (K146Q) and resulted in desumoylated THRB as the predominant form in tissues. The THRB K146Q mutant mice had normal serum thyroxine (T4), markedly elevated serum thyrotropin-stimulating hormone (TSH; 81-fold above control), and enlargement of both the pituitary and the thyroid gland. The marked elevation in TSH, despite a normal serum T4, indicated blunted feedback regulation of TSH. The THRB K146Q mutation altered the recruitment of transcription factors to the TSHβ gene promoter, compared with WT, in hyperthyroidism and hypothyroidism. Thyroid hormone content (T4, T3, and rT3) in the thyroid gland of the THRB K146Q mice was 10-fold lower (per gram tissue) than control, despite normal TSH bioactivity. The expression of thyroglobulin and dual oxidase 2 genes in the thyroid was reduced and associated with modifications of cAMP response element–binding protein DNA binding and cofactor interactions in the presence of the desumoylated THRB. Therefore, thyroid hormone production had both TSH-dependent and TSH-independent components. We conclude that THRB sumoylation at K146 was required for normal TSH feedback regulation and TH synthesis in the thyroid gland, by a TSH-independent pathway.

Biochemical Characterization and Structural Insight into Interaction and Conformation Mechanisms of Serratia marcescens Lysine Decarboxylase (SmcadA).

Inducible lysine decarboxylases (LDCs) are essential in various cellular processes of microorganisms and plants, especially under acid stress, which induces the expression of genes encoding LDCs. In this study, a novel Serratia marcesenes LDC (SmcadA) was successfully expressed in E. coli, purified and characterized. The protein had an optimal pH of 6 and a temperature of 40 °C and phylogenetic analysis to determine the evolution of SmcadA, which revealed a close relation to Enterobacteriaceae, Klebsiella sp., among others. The molecular weight of SmcadA was approximately 75 kDa after observation on SDS-PAGE and structural modeling showed the protein as a decamer, comprised of five interlinked dimers. The biocatalytic activity of the purified wild-type SmcadA (WT) was improved through site directed mutations and the results showed that the Arg595Lys mutant had the highest specific activity of 286.55 U/mg, while the Ser512Ala variant and wild-type SmcadA had 215.72 and 179.01 U/mg, respectively. Furthermore, molecular dynamics simulations revealed that interactions through hydrogen bonds between the protein residues and cofactor pyridoxal-5-phosphate (PLP) are vital for biocatalysis. Molecular Dynamics (MD) simulations also indicated that mutations conferred structural changes on protein residues and PLP hence altered the interacting residues with the cofactor, subsequently influencing substrate bioconversion. Moreover, the temperature also induced changes in orientation of cofactor PLP and amino acid residues. This work therefore demonstrates the successful expression and characterization of the purified novel lysine decarboxylase from Serratia marcesenes and provided insight into the mechanism of protein–cofactor interactions, highlighting the role of protein–ligand interactions in altering cofactor and binding site residue conformations, thus contributing to improved biocatalysis.

Recent Advances in Chemical Biology Using Benzophenones and Diazirines as Radical Precursors.

The use of light-activated chemical probes to study biological interactions was first discovered in the 1960s, and has since found many applications in studying diseases and gaining deeper insight into various cellular mechanisms involving protein–protein, protein–nucleic acid, protein–ligand (drug, probe), and protein–co-factor interactions, among others. This technique, often referred to as photoaffinity labelling, uses radical precursors that react almost instantaneously to yield spatial and temporal information about the nature of the interaction and the interacting partner(s). This review focuses on the recent advances in chemical biology in the use of benzophenones and diazirines, two of the most commonly known light-activatable radical precursors, with a focus on the last three years, and is intended to provide a solid understanding of their chemical and biological principles and their applications.

CANDOCK: Chemical Atomic Network-Based Hierarchical Flexible Docking Algorithm Using Generalized Statistical Potentials.

Small-molecule docking has proven to be invaluable for drug design and discovery. However, existing docking methods have several limitations such as improper treatment of the interactions of essential components in the chemical environment of the binding pocket (e.g., cofactors, metal ions, etc.), incomplete sampling of chemically relevant ligand conformational space, and the inability to consistently correlate docking scores of the best binding pose with experimental binding affinities. We present CANDOCK, a novel docking algorithm, that utilizes a hierarchical approach to reconstruct ligands from an atomic grid using graph theory and generalized statistical potential functions to sample biologically relevant ligand conformations. Our algorithm accounts for protein flexibility, solvent, metal ions, and cofactor interactions in the binding pocket that are traditionally ignored by current methods. We evaluate the algorithm on the PDBbind, Astex, and PINC proteins to show its ability to reproduce the binding mode of the ligands that is independent of the initial ligand conformation in these benchmarks. Finally, we identify the best selector and ranker potential functions such that the statistical score of the best selected docked pose correlates with the experimental binding affinities of the ligands for any given protein target. Our results indicate that CANDOCK is a generalized flexible docking method that addresses several limitations of current docking methods by considering all interactions in the chemical environment of a binding pocket for correlating the best-docked pose with biological activity. CANDOCK along with all structures and scripts used for benchmarking is available at https://github.com/chopralab/candock_benchmark.

Peptide-Based Biosensing of Redox-Active Protein-Heme Complexes Indicates Novel Mechanism for Tumor Survival under Oxidative Stress.

Signal response of several relevant protein-cofactor interactions, united in one bioassay, may greatly enhance the ability to study the intriguing molecular mechanisms of pathological process such as the tumor immunological process of chronic inflammation and oxidative stress. Here, a peptide-based multiplexed bioassay has been developed and applied in studying the interactions among ferritin, p53, and heme under oxidative stress. In a malignant breast cancer cell line, it can be observed that oxidative stress-triggered nuclear co-translocations of heme and ferritin may lead to direct molecular contact of ferritin with p53, to pass heme to p53, which subsequently sequestered into the cytoplasm, therefore forming a possible new route of tumor survival under oxidative stress, by using the stress to circumvent oxidative stress-induced apoptosis. The observed peroxidase-like activity of ferritin-heme and p53-heme complexes may also contribute to survival. Such activity is observed most prominently in triple negative or the most malignant breast cancer subtype. These results may suggest the possible future use of this bioassay in furthering the understanding of tumor molecular pathology, as well as the early detection, diagnosis, and prognosis of cancer.

Long-Lived Hydrated FMN Radicals: EPR Characterization and Implications for Catalytic Variability in Flavoproteins.

Until now, FMN/FAD radicals could not be stabilized in aqueous solution or other protic solvents because of rapid and efficient dismutation reactions. In this contribution, a novel system for stabilizing flavin radicals in aqueous solution is reported. Subsequent to trapping FMN in an agarose matrix, light-generated FMN radicals could be produced that were stable for days even under aerobic conditions, and their concentrations were high enough for extensive EPR characterization. All large hyperfine couplings could be extracted by using a combination of continuous-wave EPR and low-temperature ENDOR spectroscopy. To map differences in the electronic structure of flavin radicals, two exemplary proton hyperfine couplings were compared with published values from various neutral and anionic flavoprotein radicals: C(6)H and C(8α)H 3. It turned out that FMN•- in an aqueous environment shows the largest hyperfine couplings, whereas for FMNH• under similar conditions, hyperfine couplings are at the lower end and the values of both vary by up to 30%. This finding demonstrates that protein-cofactor interactions in neutral and anionic flavoprotein radicals can alter their electron spin density in different directions. With this aqueous system that allows the characterization of flavin radicals without protein interactions and that can be extended by using selective isotope labeling, a powerful tool is now at hand to quantify interactions in flavin radicals that modulate the reactivity in different flavoproteins.

The plasticity of redox cofactors: from metalloenzymes to redox-active DNA

Metal cofactors considerably widen the catalytic space of naturally occurring enzymes whose specific and enantioselective catalytic activity constitutes a blueprint for economically relevant chemical syntheses. To optimize natural enzymes and uncover novel reactivity, we need a detailed understanding of cofactor–protein interactions, which can be challenging to obtain in the case of enzymes with sophisticated cofactors. As a case study, we summarize recent research on the [FeFe]-hydrogenases, which interconvert protons, electrons and dihydrogen at a unique iron-based active site. We can now chemically synthesize the complex cofactor and incorporate it into an apo-protein to afford functional enzymes. By varying both the cofactor and the polypeptide components, we have obtained detailed knowledge on what is required for a metal cluster to process H2. In parallel, the design of artificial proteins and catalytically active nucleic acids are advancing rapidly. In this Perspective, we introduce these fields and outline how chemists and biologists can use this knowledge to develop novel tailored semisynthetic catalysts. The study of [FeFe]-hydrogenases exemplifies how one can manipulate even sophisticated metal clusters to afford insights into structure–function relationships of biological catalysts. This Perspective describes developments in designing artificial proteins and catalytically active nucleic acids towards minimalistic and robust semi-biological catalysts for chemical synthesis.

Functional Regulation of PPARs through Post-Translational Modifications.

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily and they are essential regulators of cell differentiation, tissue development, and energy metabolism. Given their central roles in sensing the cellular metabolic state and controlling metabolic homeostasis, PPARs became important targets of drug development for the management of metabolic disorders. The function of PPARs is mainly regulated through ligand binding, which induces structural changes, further affecting the interactions with co-activators or co-repressors to stimulate or inhibit their functions. In addition, PPAR functions are also regulated by various Post-translational modifications (PTMs). These PTMs include phosphorylation, SUMOylation, ubiquitination, acetylation, and O-GlcNAcylation, which are found at numerous modification sites. The addition of these PTMs has a wide spectrum of consequences on protein stability, transactivation function, and co-factor interaction. Moreover, certain PTMs in PPAR proteins have been associated with the status of metabolic diseases. In this review, we summarize the PTMs found on the three PPAR isoforms PPARα, PPARβ/δ, and PPARγ, and their corresponding modifying enzymes. We also discuss the functional roles of these PTMs in regulating metabolic homeostasis and provide a perspective for future research in this intriguing field.

1H magic-angle spinning NMR evolves as a powerful new tool for membrane proteins

Building on a decade of continuous advances of the community, the recent development of very fast (60 kHz and above) magic-angle spinning (MAS) probes has revolutionised the field of solid-state NMR. This new spinning regime reduces the 1H-1H dipolar couplings, so that direct detection of the larger magnetic moment available from 1H is now possible at high resolution, not only in deuterated molecules but also in fully-protonated substrates. Such capabilities allow rapid “fingerprinting” of samples with a ten-fold reduction of the required sample amounts with respect to conventional approaches, and permit extensive, robust and expeditious assignment of small-to-medium sized proteins (up to ca. 300 residues), and the determination of inter-nuclear proximities, relative orientations of secondary structural elements, protein-cofactor interactions, local and global dynamics.Fast MAS and 1H detection techniques have nowadays been shown to be applicable to membrane-bound systems. This paper reviews the strategies underlying this recent leap forward in sensitivity and resolution, describing its potential for the detailed characterization of membrane proteins.

Proton-Based Structural Analysis of a Heptahelical Transmembrane Protein in Lipid Bilayers.

The structures and properties of membrane proteins in lipid bilayers are expected to closely resemble those in native cell-membrane environments, although they have been difficult to elucidate. By performing solid-state NMR measurements at very fast (100 kHz) magic-angle spinning rates and at high (23.5 T) magnetic field, severe sensitivity and resolution challenges are overcome, enabling the atomic-level characterization of membrane proteins in lipid environments. This is demonstrated by extensive 1H-based resonance assignments of the fully protonated heptahelical membrane protein proteorhodopsin, and the efficient identification of numerous 1H–1H dipolar interactions, which provide distance constraints, inter-residue proximities, relative orientations of secondary structural elements, and protein–cofactor interactions in the hydrophobic transmembrane regions. These results establish a general approach for high-resolution structural studies of membrane proteins in lipid environments via solid-state NMR.

Vibrational Probes of Molybdenum Cofactor-Protein Interactions in Xanthine Dehydrogenase.

The pyranopterin dithiolene (PDT) ligand is an integral component of the molybdenum cofactor (Moco) found in all molybdoenzymes with the sole exception of nitrogenase. However, the roles of the PDT in catalysis are still unknown. The PDT is believed to be bound to the proteins by an extensive hydrogen-bonding network, and it has been suggested that these interactions may function to fine-tune Moco for electron- and atom-transfer reactivity in catalysis. Here, we use resonance Raman (rR) spectroscopy to probe Moco-protein interactions using heavy-atom congeners of lumazine, molecules that bind tightly to both wild-type xanthine dehydrogenase (wt-XDH) and its Q102G and Q197A variants following enzymatic hydroxylation to the corresponding violapterin product molecules. The resulting enzyme-product complexes possess intense near-IR absorption, allowing high-quality rR spectra to be collected on wt-XDH and the Q102G and Q197A variants. Small negative frequency shifts relative to wt-XDH are observed for the low-frequency Moco vibrations. These results are interpreted in the context of weak hydrogen-bonding and/or electrostatic interactions between Q102 and the -NH2 terminus of the PDT, and between Q197 and the terminal oxo of the Mo≡O group. The Q102A, Q102G, Q197A, and Q197E variants do not appreciably affect the kinetic parameters kred and kred/KD, indicating that a primary role for these glutamine residues is to stabilize and coordinate Moco in the active site of XO family enzymes but to not directly affect the catalytic throughput. Raman frequency shifts between wt-XDH and its Q102G variant suggest that the changes in the electron density at the Mo ion that accompany Mo oxidation during electron-transfer regeneration of the catalytically competent active site are manifest in distortions at the distant PDT amino terminus. This implies a primary role for the PDT as a conduit for facilitating enzymatic electron-transfer reactivity in xanthine oxidase family enzymes.

In Silico Approaches to Identify Mutagenesis Targets to Probe and Alter Protein-Cofactor and Protein-Protein Functional Relationships.

When performing site-directed mutagenesis experiments to study protein structure-function relationships, ideally one would know the structure of the protein under study. It is also very useful to have structures of multiple related proteins in order to determine whether or not particular amino acid residues are conserved in the structures either in the active site of an enzyme at the surface of a protein or at a putative protein-protein interface. While many protein structures are available in the Protein Data Base (PDB), a structure of the protein of interest may not be available. In the study of reversible and often transient protein-protein interactions it is rare to have a structure of the complex of the two interacting proteins. In this chapter, methods are described for comparing protein structures, generating putative structures of proteins with homology models based on the protein primary sequence, and generating docking models to predict interaction sites between proteins and cofactor-protein interactions. The rationale used to predict mutagenesis targets from these structures and models is also described.

Analytical Methods for Assessing the Effects of Site-Directed Mutagenesis on Protein-Cofactor and Protein-Protein Functional Relationships.

To completely understand the role of an amino acid residue that is targeted for site-directed mutagenesis a thorough analysis of the impact that the mutation has on the function of the protein is required. General methods for performing site-directed mutagenesis and expressing the recombinant protein variant are described. Protein-cofactor interactions are important because cofactors are often directly involved in facilitating catalysis by enzymes and in electron transfer by redox proteins. Many cofactors also have characteristic spectroscopic properties. As such, general methods are described to analyze the spectroscopic, redox and catalytic properties of protein-bound cofactors. Methods for assessing the effects of a mutation on protein-protein interactions are also described. Lastly, methods for assessing the overall structural integrity of the protein are described, as this is important to ensure that the mutation has not caused a global disruption of protein structure, rather than a specific effect on function.

A network analysis of cofactor-protein interactions for analyzing associations between human nutrition and diseases.

The involvement of vitamins and other micronutrients in intermediary metabolism was elucidated in the mid 1900’s at the level of individual biochemical reactions. Biochemical pathways remain the foundational knowledgebase for understanding how micronutrient adequacy modulates health in all life stages. Current daily recommended intakes were usually established on the basis of the association of a single nutrient to a single, most sensitive adverse effect and thus neglect interdependent and pleiotropic effects of micronutrients on biological systems. Hence, the understanding of the impact of overt or sub-clinical nutrient deficiencies on biological processes remains incomplete. Developing a more complete view of the role of micronutrients and their metabolic products in protein-mediated reactions is of importance. We thus integrated and represented cofactor-protein interaction data from multiple and diverse sources into a multi-layer network representation that links cofactors, cofactor-interacting proteins, biological processes, and diseases. Network representation of this information is a key feature of the present analysis and enables the integration of data from individual biochemical reactions and protein-protein interactions into a systems view, which may guide strategies for targeted nutritional interventions aimed at improving health and preventing diseases.

Abstract 2448: Towards structure-based drug design of 3-phosphoglycerate dehydrogenase inhibitors

Abstract Background The NAD+-requiring enzyme 3-phosphoglycerate dehydrogenase (PHGDH) diverts glycolytic flux into serine production and folate metabolism by catalyzing the oxidization of 3-phosphoglycerate to phosphohydroxypyruvate. The PHGDH gene is located on chromosome 1(1p12), a region frequently amplified in melanoma and certain breast cancer forms. PHGDH knockdown in cells with amplified PHGDH or overexpressing PHGDH at the protein level resulted in cell growth inhibition. In addition ectopic overexpression of PHGDH in a non-tumorigenic cell line induced morphological changes characteristic of transformation. Therefore, inhibitors of PHGDH may be therapeutically valuable. No PHGDH inhibitors have been reported to date, our aim is to develop PHGDH inhibitors targeting the cofactor (and substrate) binding site. Methods Full length PHGDH and the catalytic subunit (sPHGDH) were expressed in, and isolated from E. coli. PHGDH was found to co-purify with its cofactor, making it necessary to abolish the cofactor-protein interaction to investigate inhibitor binding at the co-factor site. To accomplish this, site-directed sPHGDH mutants were constructed and purified. To characterize the mutants, their enzymatic activity and ability to bind to NADH (assessed by thermal stability and isothermal titration calorimetry (ITC)) were measured, and their structures were characterized by circular dichroism (CD) spectroscopy and on-going x-ray crystallographic studies. Results sPHGDH retained 60% of the enzymatic activity of the full length protein whereas introduction of single or double point mutations around the cofactor or substrate binding site resulted in complete abolition of enzymatic activity. The binding of NADH (1mM) to PHGDH and sPHGDH resulted in increases in melting temperature (ΔTm) of 8.4 ± 0.2 °C and 9.3 ± 0.5 °C respectively. Mutation of the cofactor binding site resulted in reduced ability to bind NADH (ΔTm = 0.8 ± 0.2 °C, 2.3 ± 0.1 °C) whereas mutation at the substrate binding site, directly adjacent to the cofactor binding site, had no significant effect on the ability to bind NADH. Congruent findings were obtained via ITC determining a binding affinity (Kd) of 0.66 μM for wt sPHGDH and a reduced binding affinity of 3-30 fold for the mutated proteins. CD measurements showed that the proteins mutated at the co-factor binding site had undergone minor changes in the secondary structure compared to the wt sPHGDH whereas mutation at the substrate binding site had no effect on the secondary structure elements. Conclusion Although the mutations carried out were single or double point mutations only, cofactor binding could be reduced substantially, giving rise to markedly lower catalytic activity. These investigations illustrate a promising way of making proteins with large, tight binding ligands accessible to structure-based drug-design. Citation Format: Judith E. Unterlass, Nabila Aljufri, Sophie Bex, Celine Cano, Martin E M Noble, Nicola J. Curtin. Towards structure-based drug design of 3-phosphoglycerate dehydrogenase inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2448. doi:10.1158/1538-7445.AM2015-2448

Sumo‐modification of pxr alters protein cofactor interactions (1062.11)

Pregnane X Receptor (PXR, NR1I2) is a ligand‐activated nuclear receptor superfamily member expressed at high levels within the entero‐hepatic system in mammals. The primary function of PXR is to regulate the metabolism and transport of xenobiotics and endogenous compounds by controlling the expression of genes encoding major drug metabolizing enzymes and drug transporter proteins. To mediate its function PXR interacts with a plethora of classical co‐regulatory proteins including, but not limited to, Nuclear Receptor Co‐Repressors (NCoR1/NCoR2), Steroid Receptor Co‐activator proteins (SRC‐1/SRC‐2/SRC‐3) and p300/CBP co‐integrator proteins. SUMOylation is a reversible post‐translational modification that plays a central role in regulating multiple cellular activities such as protein‐protein interactions. While direct PXR‐target gene activation programs are currently well characterized, little is known about SUMO‐modification of PXR. Our working hypothesis is that SUMO‐modified PXR alters its protein‐protein interaction profile. Thus, we have created a GAL4‐PXR fusion system that allows us to directly examine SUMO‐PXR interaction with the aforementioned PXR‐binding proteins. Future efforts are focused on developing novel methods for identification of SUMO‐PXR‐binding proteins using non‐biased methods.Grant Funding Source: Supported by NIH DK090558

Active and silent chromophore isoforms for phytochrome Pr photoisomerization: An alternative evolutionary strategy to optimize photoreaction quantum yields.

Photoisomerization of a protein bound chromophore is the basis of light sensing of many photoreceptors. We tracked Z-to-E photoisomerization of Cph1 phytochrome chromophore PCB in the Pr form in real-time. Two different phycocyanobilin (PCB) ground state geometries with different ring D orientations have been identified. The pre-twisted and hydrogen bonded PCBa geometry exhibits a time constant of 30 ps and a quantum yield of photoproduct formation of 29%, about six times slower and ten times higher than that for the non-hydrogen bonded PCBb geometry. This new mechanism of pre-twisting the chromophore by protein-cofactor interaction optimizes yields of slow photoreactions and provides a scaffold for photoreceptor engineering.

A Simple Method to Measure Protein Side-Chain Mobility Using NMR Chemical Shifts

Protein side-chain motions are involved in many important biological processes including enzymatic catalysis, allosteric regulation, and the mediation of protein-protein, protein-DNA, protein-RNA, and protein-cofactor interactions. NMR spectroscopy has long been used to provide insights into the motions of side-chain groups. Currently, the method of choice for studying side-chain dynamics by NMR is the measurement of methyl (2)H autorelaxation. Methyl (2)H autorelaxation exhibits simple relaxation mechanisms and can be straightforwardly converted to meaningful dynamic parameters. However, methyl groups can only be found in 6 of 19 side-chain bearing amino acids. Consequently, only a sparse assessment of protein side-chain dynamics is possible. Therefore, there is a significant interest in developing novel methods of studying side-chain motions that can be applied to all types of side-chains. Here, we show how side-chain chemical shifts can be used to determine the magnitude of fast side-chain motions in proteins. The chemical shift method is applicable to all side-chain bearing residues and does not require any additional measurements beyond standard NMR experiments for backbone and side-chain assignments.