Utility of fluorescent heme analogue ZnPPIX to monitor conformational heterogeneity in vertebrate hexa-coordinated globins

Metallomics ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 906-913 ◽  
Author(s):  
Antonija Tangar ◽  
Valérie Derrien ◽  
Ruipeng Lei ◽  
Maria Jose Santiago Estevez ◽  
Pierre Sebban ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Structural Heterogeneity ◽  
Conformational Heterogeneity ◽  
Heme Binding ◽  
Fluorescent Analogues

Fluorescent analogues of cytoglobin and neuroglobin reveal an increased structural heterogeneity of the heme binding pocket.

Study on the Gas Phase Stability of Heme-binding Pocket in Cytochrome Tb5 and Its Mutants by Electrospray Mass Spectrometry

2010 ◽  
Vol 20 (12) ◽  
pp. 1540-1545 ◽  
Author(s):  
Chong-Tian Yu ◽  
Yin-Long Guo ◽  
Long Lü ◽  
Yun-Hua Wang ◽  
Ping Yao ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Phase Stability ◽  
Electrospray Mass Spectrometry ◽  
Binding Pocket ◽  
Heme Binding

Two Stacked Heme Molecules in the Binding Pocket of the Periplasmic Heme-Binding Protein HmuT from Yersinia pestis

2010 ◽  
Vol 404 (2) ◽  
pp. 220-231 ◽  
Author(s):  
Daniel Mattle ◽  
Antra Zeltina ◽  
Jae-Sung Woo ◽  
Birke A. Goetz ◽  
Kaspar P. Locher
Keyword(s):  
Yersinia Pestis ◽  
Binding Protein ◽  
Binding Pocket ◽  
Heme Binding ◽  
Heme Binding Protein

scholarly journals Conformational heterogeneity of Savinase from NMR, HDX-MS and X-ray diffraction analysis

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9408
Author(s):  
Shanshan Wu ◽  
Tam T.T.N. Nguyen ◽  
Olga V. Moroz ◽  
Johan P. Turkenburg ◽  
Jens E. Nielsen ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Solvent Accessibility ◽  
Conformational Dynamics ◽  
Structural Heterogeneity ◽  
Catalytic Process ◽  
Conformational Heterogeneity ◽  
X Ray ◽  
Exchange Mass Spectrometry ◽  
Bacillus Lentus ◽  
Hdx Ms

Background Several examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme resemble the conformations of intermediate states in the catalytic process. Previous work on the subtilisin protease, Savinase, from Bacillus lentus by NMR spectroscopy suggested that this enzyme undergoes slow conformational dynamics around the substrate binding site. However, the functional importance of such dynamics is unknown. Methods Here we have probed the conformational heterogeneity in Savinase by following the temperature dependent chemical shift changes. In addition, we have measured changes in the local stability of the enzyme when the inhibitor phenylmethylsulfonyl fluoride is bound using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Finally, we have used X-ray crystallography to compare electron densities collected at cryogenic and ambient temperatures and searched for possible low populated alternative conformations in the crystals. Results The NMR temperature titration shows that Savinase is most flexible around the active site, but no distinct alternative states could be identified. The HDX shows that modification of Savinase with inhibitor has very little impact on the stability of hydrogen bonds and solvent accessibility of the backbone. The most pronounced structural heterogeneities detected in the diffraction data are limited to alternative side-chain rotamers and a short peptide segment that has an alternative main-chain conformation in the crystal at cryo conditions. Collectively, our data show that there is very little structural heterogeneity in the resting state of Savinase and hence that Savinase does not rely on conformational selection to drive the catalytic process.

scholarly journals Structural heterogeneity in biliverdin modulates spectral properties of Sandercyanin fluorescent protein

2021 ◽  
Author(s):  
Swagatha Ghosh ◽  
Sayan Mondal ◽  
Keerti Yadav ◽  
Shantanu Aggarwal ◽  
Wayne F. Schaefer ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Spectral Properties ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Binding Pocket ◽  
Structural Heterogeneity ◽  
Wild Type ◽  
Absorbing States

Sandercyanin, a blue homo-tetrameric lipocalin protein purified from Canadian walleye (Stizostedion vitreus), is the first far-red fluorescent protein reported in vertebrates. Sandercyanin binds non-covalently to biliverdin IXα (BLA) and fluoresces at 675nm on excitation at 375nm and 635nm. Sandercyanin fluorescence can be harnessed for many in vivo applications when engineered into a stable monomeric form. Here, we report the spectral properties and crystal structures of engineered monomeric Sandercyanin-BLA complexes. Compared to wild-type protein, monomeric Sandercyanin (~18kDa) binds BLA with similar affinities and show a broad red- shifted absorbance spectra but possess reduced quantum efficiency. Crystal structures reveal D-ring pyrrole of BLA rotated around the C14-C15 bond, which is stabilized by neighboring aromatic residues and increased water-mediated polar contacts in the BLA-binding pocket. A tetrameric Sandercyanin variant (Tyr-142-Ala) co-displaying red- and far-red absorbing states, and reduced fluorescence shows similar conformational changes in BLA binding pocket. Our results suggest that D-ring flexibility of BLA and its rearrangement reduces the fluorescence quantum-yield of monomeric Sandercyanin. Structures of monomeric Sandercyanin could be utilized as prototypes to generate bright BLA-inducible fluorescent proteins. Further, our study postulates a mechanism for modulating photo-states in BLA- bound lipocalins, known only in phytochromes till date.

scholarly journals Photosynthetic Water Oxidation in Cytochromeb559Mutants Containing a Disrupted Heme-binding Pocket

2001 ◽  
Vol 276 (34) ◽  
pp. 31986-31993 ◽  
Author(s):  
Francisco Morais ◽  
Kristina Kühn ◽  
David H. Stewart ◽  
James Barber ◽  
Gary W. Brudvig ◽  
...  
Keyword(s):  
Water Oxidation ◽  
Binding Pocket ◽  
Heme Binding ◽  
Photosynthetic Water Oxidation

scholarly journals An Induced Fit Conformational Change Underlies the Binding Mechanism of the Heme Transport Proteobacteria-Protein HemS

2006 ◽  
Vol 281 (43) ◽  
pp. 32606-32610 ◽  
Author(s):  
Sabine Schneider ◽  
Katherine H. Sharp ◽  
Paul D. Barker ◽  
Max Paoli
Keyword(s):  
Conformational Changes ◽  
Structural Data ◽  
Binding Pocket ◽  
Bound State ◽  
Uptake System ◽  
Heme Binding ◽  
Binding Mechanism ◽  
Induced Fit ◽  
Heme Transport ◽  
Β Sheet

Bacteria rely on their environment and/or host to acquire iron and have evolved specialized systems to sequester and transport heme. The heme uptake system HemRSTUV is common to proteobacteria, and a major challenge is to understand the molecular mechanism of heme binding and transfer between the protein molecules that underlie this heme transport relay process. In the Gram-negative pathogen Yersinia enterocolitica, the HemRSTUV system culminates with the cytoplasmic recipient HemS, which stores and delivers heme for cellular needs. HemS belongs to a family of proteins essential and unique to proteobacteria. Here we report on the binding mechanism of HemS based on structural data from its apo- and ligand-loaded forms. This heme carrier protein associates with its cargo through a novel, partly preformed binding pocket, formed between a large β-sheet dome and a three-helix subdomain. In addition to a histidine interacting with the iron, the complex is stabilized by a distal non-coordinating arginine that packs along the porphyrin plane and extensive electrostatic contacts that firmly anchor the heme propionate groups within the protein. Comparison of apo- and ligand-bound HemS crystal structures reveals striking conformational changes that underlie a “heme-induced fit” binding mechanism. Local shifts in amino acid positions combine with global, rigid body-like domain movements, and together, these bring about a switch from an open, apo-form to a closed, bound state. This is the first report in which both liganded and unliganded forms of a heme transport protein are described, thus providing penetrating insights into its mechanism of heme binding and release.

Spectroscopic characterization of the heme binding (GAF) domain of two sensor kinases from Methanosarcina acetivorans

2019 ◽  
Vol 23 (07n08) ◽  
pp. 930-942
Author(s):  
Kerstin Fiege ◽  
Christian Twittenhoff ◽  
Kathrin Kwiatkowski ◽  
Nicole Frankenberg-Dinkel
Keyword(s):  
Spectroscopic Characterization ◽  
Binding Pocket ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Heme Binding ◽  
Methanosarcina Acetivorans ◽  
Vis Spectroscopy ◽  
Sensor Kinases ◽  
Covalently Linked ◽  
Heme Cofactor

The sensor kinases MsmS and RdmS from the methanogenic archaeon Methanosarcina acetivorans are multidomain proteins containing a covalently linked heme cofactor. This cofactor is connected via a single cysteine residue in a GAF domain. Although both proteins were shown to display a redox-dependent control of the downstream kinase module, this property appears to be independent of the heme cofactor. We therefore envision an additional sensor role for the heme cofactor. In order to learn more about the heme binding pocket and its constitution, UV-vis spectroscopy in combination with site-directed mutagenesis was performed on the isolated heme-binding sGAF2 domain and the full-length protein. The data indicate a 6-coordinated heme with a proximal histidine ligand and a smaller ligand, likely a water molecule on the distal site. The latter is also thought to be the sensory site and is shown to easily undergo ligand exchange.

scholarly journals Precision design of single and multi-heme de novo proteins

2020 ◽  
Author(s):  
George H. Hutchins ◽  
Claire E. M. Noble ◽  
Hector Blackburn ◽  
Ben Hardy ◽  
Charles Landau ◽  
...  
Keyword(s):  
Crystal Structure ◽  
De Novo ◽  
Molten Globule ◽  
Computational Design ◽  
Binding Pocket ◽  
Oxygen Binding ◽  
Heme Binding ◽  
Functional Protein ◽  
Precision Design ◽  
Protein Functions

AbstractThe de novo design of simplified porphyrin-binding helical bundles is a versatile approach for the construction of valuable biomolecular tools to both understand and enhance protein functions such as electron transfer, oxygen binding and catalysis. However, the methods utilised to design such proteins by packing hydrophobic side chains into a buried binding pocket for ligands such as heme have typically created highly flexible, molten globule-like structures, which are not amenable to structural determination, hindering precise engineering of subsequent designs. Here we report the crystal structure of a de novo two-heme binding “maquette” protein, 4D2, derived from the previously designed D2 peptide, offering new opportunities for computational design and re-engineering. The 4D2 structure was used as a basis to create a range of heme binding proteins which retain the architecture and stability of the initial crystal structure. A well-structured single-heme binding variant was constructed by computational sequence redesign of the hydrophobic protein core, assessed by NMR, and utilised for experimental validation of computational redox prediction and design. The structure was also extended into a four-heme binding helical bundle resembling a molecular wire. Despite a molecular weight of only 24kDa, imaging by CryoEM illustrated a remarkable level of detail in this structure, indicating the positioning of both the secondary structure and the heme cofactors. The design and determination of atomic-level resolution in such de novo proteins is an invaluable resource for the continued development of novel and functional protein tools.

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