Determination of Myoglobin Stability by Circular Dichroism Spectroscopy: Classic and Modern Data Analysis

Mehl, Andrew F.; Crawford, Mary Ann; Zhang, Lei

doi:10.1021/ed086p600

Cited by 22 publications

(30 citation statements)

References 4 publications

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“…Thermodynamic stability parameters of HepI-WT and mutants were calculated as previously reported by Mehl et.al, using ellipticities at 211 and 222 nm (Supplemental Table S3). 20 Percent composition of secondary structure (α-helicity and β-strand percentages) was determined using the K2D3 CD spectral analysis tool (http://cbdm-01.zdv.uni-mainz.de/~andrade/k2d3//info/about.html). …”

Section: Methodsmentioning

confidence: 99%

The Stories Tryptophans Tell: Exploring Protein Dynamics of Heptosyltransferase I from Escherichia coli

et al. 2017

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Heptosyltransferase I (HepI) catalyzes the addition of L-glycero-β-D-manno-heptose onto Kdo2-Lipid A, as part of the biosynthesis of the core region of lipopolysaccharide (LPS). Gram-negative bacteria with gene knockouts of HepI have reduced virulence and enhanced susceptibility to hydrophobic antibiotics, making design of inhibitors against HepI of interest. Since HepI protein dynamics are partially rate-limiting, disruption of protein dynamics might provide a new strategy for inhibiting HepI. Discerning the global mechanism of HepI is anticipated to aid development of inhibitors of LPS biosynthesis. Herein, dynamic protein rearrangements involved in the HepI catalytic cycle were probed by combining mutagenesis with intrinsic tryptophan fluorescence and circular dichroism analyses. Using wild-type and mutant forms of HepI, multiple dynamic regions were identified via monitoring fluorescence wavelength changes. Interestingly, Trp residues (Trp199 and Trp217) in the C-terminal domain (which binds ADP-Heptose) are in a more hydrophobic environment upon ODLA binding to the N-terminal domain. These residues are adjacent to the ADP-Heptose binding site (with Trp217 in van der Waals contact with the Adenine ring of ADP-Heptose), suggesting that the two binding sites interact to report on the occupancy state of the enzyme. ODLA binding was also accompanied by a significant stabilization of HepI (heating to 95 °C fails to denature the protein when in the presence of ODLA). These results suggest that conformational rearrangements, from an induced fit model of substrate binding to HepI, are important for catalysis, and the disruption of these dynamics may serve as novel mechanism for inhibiting this and other glycosyltransferase enzymes.

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Section: Methodsmentioning

confidence: 99%

The Stories Tryptophans Tell: Exploring Protein Dynamics of Heptosyltransferase I from Escherichia coli

et al. 2017

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“…At physiological conditions, HoloMb is stable by typically 45–55 kJ/mol (with higher stability observed for Mbs of diving mammals [12] , [13] ). It unfolds with a barrier of ∼45 kJ/mol (horse Mb) [14] with T m ∼80–85°C [11] , a process that, in contrast to many other small, single-domain proteins that fold by two-state processes [15] , may involve several intermediates (I) between the native (N) and unfolded (U) states [16] . Loss of heme takes days under physiological conditions due to the very high protein-heme affinity [17] , with a rate of heme loss of ∼0.01 h −1 [18] .…”

Section: Introductionmentioning

confidence: 99%

Unfolding Simulations of Holomyoglobin from Four Mammals: Identification of Intermediates and β-Sheet Formation from Partially Unfolded States

Dasmeh

Kepp

2013

PLoS ONE

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Myoglobin (Mb) is a centrally important, widely studied mammalian protein. While much work has investigated multi-step unfolding of apoMb using acid or denaturant, holomyoglobin unfolding is poorly understood despite its biological relevance. We present here the first systematic unfolding simulations of holoMb and the first comparative study of unfolding of protein orthologs from different species (sperm whale, pig, horse, and harbor seal). We also provide new interpretations of experimental mean molecular ellipticities of myoglobin intermediates, notably correcting for random coil and number of helices in intermediates. The simulated holoproteins at 310 K displayed structures and dynamics in agreement with crystal structures (R g ∼1.48–1.51 nm, helicity ∼75%). At 400 K, heme was not lost, but some helix loss was observed in pig and horse, suggesting that these helices are less stable in terrestrial species. At 500 K, heme was lost within 1.0–3.7 ns. All four proteins displayed exponentially decaying helix structure within 20 ns. The C- and F-helices were lost quickly in all cases. Heme delayed helix loss, and sperm whale myoglobin exhibited highest retention of heme and D/E helices. Persistence of conformation (RMSD), secondary structure, and ellipticity between 2–11 ns was interpreted as intermediates of holoMb unfolding in all four species. The intermediates resemble those of apoMb notably in A and H helices, but differ substantially in the D-, E- and F-helices, which interact with heme. The identified mechanisms cast light on the role of metal/cofactor in poorly understood holoMb unfolding. We also observed β-sheet formation of several myoglobins at 500 K as seen experimentally, occurring after disruption of helices to a partially unfolded, globally disordered state; heme reduced this tendency and sperm-whale did not display any sheet propensity during the simulations.

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“…Figure 8 shows the CD spectra of commercial and extracted myoglobin from human serum. As can be seen from the figure, bands between 190 and 230 nm show the α -helical type of secondary structure of 500 mg/mL myoglobin (blue spectrum) [ 44 , 45 ]. The red spectrum showed that α -helical type of secondary structure in 208-222 nm interval remained for isolated myoglobin.…”

Section: Resultsmentioning

confidence: 99%

Selective Recognition of Myoglobin in Biological Samples Using Molecularly Imprinted Polymer-Based Affinity Traps

Keçili

2018

International Journal of Analytical Chemistry

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The current work demonstrates the design, characterization, and preparation of molecularly imprinted microspheres for the selective detection of myoglobin in serum samples. The suspension polymerization approach was applied for the preparation of myoglobin imprinted microspheres. For this purpose, N-methacryloylamino folic acid-Nd3+ (MAFol- Nd3+) was chosen as the complex functional monomer. The optimization studies were performed changing the medium pH, temperature, and myoglobin concentration. pH 7.0 was determined as the optimum value where the prepared imprinted microspheres displayed maximum binding for myoglobin. The maximum binding capacity was achieved as 623 mgg−1. In addition, the selectivity studies were conducted. The results confirmed that the imprinted microspheres showed great selectivity towards myoglobin in the existence of hemoglobin, cytochrome c, and lysozyme which were chosen as potentially competing proteins.

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Determination of Myoglobin Stability by Circular Dichroism Spectroscopy: Classic and Modern Data Analysis

Cited by 22 publications

References 4 publications

The Stories Tryptophans Tell: Exploring Protein Dynamics of Heptosyltransferase I from Escherichia coli

The Stories Tryptophans Tell: Exploring Protein Dynamics of Heptosyltransferase I from Escherichia coli

Unfolding Simulations of Holomyoglobin from Four Mammals: Identification of Intermediates and β-Sheet Formation from Partially Unfolded States

Selective Recognition of Myoglobin in Biological Samples Using Molecularly Imprinted Polymer-Based Affinity Traps

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