When Less Is More: Combining Site-Specific Isotope Labeling and NMR Unravels Structural Details of Huntingtin Repeats

Abstract Site specific methyl labeling combined with methyl TROSY offers a powerful NMR approach to study structure and dynamics of proteins and protein complexes of high molecular weight. Robust and cost-effective methods have been developed for site specific protein 1H/13C methyl labeling in an otherwise deuterated background in bacteria. However, bacterial systems are not suitable for expression and isotope labeling of many eukaryotic and membrane proteins. The yeast Pichia pastoris (P. pastoris) is a commonly used host for expression of eukaryotic proteins, and site-specific methyl labeling of perdeuterated eukaryotic proteins has recently been achieved with this system. However, the practical utility of methyl labeling and deuteration in P. pastoris is limited by high costs. Here, we describe an improved method for 1H/13C-labeling of the δ-methyl group of isoleucine residues in a perdeuterated background, which reduces the cost by ≥ 50% without compromising the efficiency of isotope enrichment. We have successfully implemented this method to label actin and a G-protein coupled receptor. Our approach will facilitate studies of the structure and dynamics of eukaryotic proteins by NMR spectroscopy.

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Dissection of Hydrogen Bond Interaction Network around an Iron–Sulfur Cluster by Site-Specific Isotope Labeling of Hyperthermophilic Archaeal Rieske-Type Ferredoxin

Journal of the American Chemical Society ◽

10.1021/ja308049u ◽

2012 ◽

Vol 134 (48) ◽

pp. 19731-19738 ◽

Cited By ~ 13

Author(s):

Toshio Iwasaki ◽

Risako Fukazawa ◽

Yoshiharu Miyajima-Nakano ◽

Amgalanbaatar Baldansuren ◽

Shinichi Matsushita ◽

...

Keyword(s):

Hydrogen Bond ◽

Isotope Labeling ◽

Interaction Network ◽

Hydrogen Bond Interaction ◽

Iron Sulfur Cluster ◽

Site Specific ◽

Sulfur Cluster ◽

Iron Sulfur ◽

Bond Interaction

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Intensity Revival of Weak Symmetric Infrared Band is Possible in Ubiquinone Molecules through the Asymmetric Site-specific Isotope Labeling

Himalayan Physics ◽

10.3126/hj.v5i0.12838 ◽

2015 ◽

Vol 5 ◽

pp. 39-46

Author(s):

Hari Prasad Lamichhane

Keyword(s):

Spectral Region ◽

Isotope Labeling ◽

Spectral Band ◽

Polarizable Continuum Model ◽

Infrared Band ◽

Site Specific ◽

13C Labeling ◽

Spectral Bands ◽

Weak Intensity ◽

Ir Bands

Modifications of Infrared (IR) spectral bands of ubiquinone molecule (UQ1) upon site-specific 13C labeling at the C5 or C6 position are studied in CCl4 using Gaussian 03. Polarizable continuum model (PCM) has been used to optimize the UQ1 molecule in solvent. The unlabeled neutral ubiquinone molecule consists of three intense IR bands in the frequency region between 1700 cm-1 to 1550 cm-1. The symmetric fourth band in this spectral region does not appear in the spectrum because of very weak intensity. However, site-specific 13C labeling at C5 or C6 position removes the molecular symmetry and hence there appear four equivalent IR bands in the spectral region thus considered. This observation explains why there appears an extra spectral band in the experimental spectra observed by Brudler, R. et al [1].The Himalayan Physics Year 5, Vol. 5, Kartik 2071 (Nov 2014)Page: 39-46

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Structural analysis of proteins by isotope-edited FTIR spectroscopy

Spectroscopy An International Journal ◽

10.1155/2010/634831 ◽

2010 ◽

Vol 24 (1-2) ◽

pp. 37-43 ◽

Cited By ~ 11

Author(s):

Suren A. Tatulian

Keyword(s):

Structural Analysis ◽

Ftir Spectroscopy ◽

Structural Changes ◽

Isotope Labeling ◽

Structural Information ◽

Attenuated Total Reflection ◽

Site Specific ◽

Segmental Isotope Labeling ◽

Protein Membrane

Structure determination of multidomain proteins or protein–membrane complexes is one of the most challenging tasks in modern structural biology. High-resolution techniques, like NMR or X-ray crystallography, are limited to molecules of moderate size or those that can be crystallized easily. Both methods encounter serious technical obstacles in structural analysis of protein–membrane systems. This work describes an emerging biophysical technique that combines segmental isotope labeling of proteins with Fourier transform infrared (FTIR) spectroscopy, which provides site-specific structural information on proteins and allows structural characterization of protein–membrane complexes. Labeling of a segment of the protein with13C results in infrared spectral resolution of the labeled and unlabeled parts and thus allows identification of structural changes in specific domains/segments of the protein that accompany functional transitions. Segmental isotope labeling also allows determination of the precise configuration of protein–membrane complexes by polarized attenuated total reflection FTIR (ATR–FTIR) spectroscopy. These new developments offer solutions to functionally important site-specific structural changes in proteins and protein–membrane complexes that are hard to approach using conventional methods.

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Site-specific isotope labeling of long RNA for structural and mechanistic studies

Nucleic Acids Research ◽

10.1093/nar/gkr951 ◽

2011 ◽

Vol 40 (1) ◽

pp. e7-e7 ◽

Cited By ~ 8

Author(s):

Ikumi Kawahara ◽

Kaichiro Haruta ◽

Yuta Ashihara ◽

Daichi Yamanaka ◽

Mituhiro Kuriyama ◽

...

Keyword(s):

Isotope Labeling ◽

Mechanistic Studies ◽

Site Specific

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A Site-Specific Low-Enrichment15N,13C Isotope-Labeling Approach to Unambiguous NMR Spectral Assignments in Nucleic Acids

Journal of the American Chemical Society ◽

10.1021/ja011977m ◽

2002 ◽

Vol 124 (7) ◽

pp. 1160-1161 ◽

Cited By ~ 80

Author(s):

Anh Tuân Phan ◽

Dinshaw J. Patel

Keyword(s):

Nucleic Acids ◽

Isotope Labeling ◽

Site Specific ◽

13C Isotope ◽

Labeling Approach ◽

A Site

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Michler’s hydrol blue elucidates structural differences in prion strains

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2001732117 ◽

2020 ◽

Vol 117 (47) ◽

pp. 29677-29683

Author(s):

Yiling Xiao ◽

Sandra Rocha ◽

Catherine C. Kitts ◽

Anna Reymer ◽

Tamás Beke-Somfai ◽

...

Keyword(s):

Binding Site ◽

Conformational Changes ◽

Density Functional ◽

Amyloid Fibrils ◽

Site Specific ◽

Yeast Prions ◽

Structural Details ◽

Structural Differences ◽

Environmentally Sensitive ◽

A Site

Yeast prions provide self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that confer distinct phenotypes when introduced into cells that do not carry the prion. Classic dyes, such as thioflavin T and Congo red, exhibit large increases in fluorescence when bound to amyloids, but these dyes are not sensitive to local structural differences that distinguish amyloid strains. Here we describe the use of Michler’s hydrol blue (MHB) to investigate fibrils formed by the weak and strong prion fibrils of Sup35NM and find that MHB differentiates between these two polymorphs. Quantum mechanical time-dependent density functional theory (TDDFT) calculations indicate that the fluorescence properties of amyloid-bound MHB can be correlated to the change of binding site polarity and that a tyrosine to phenylalanine substitution at a binding site could be detected. Through the use of site-specific mutants, we demonstrate that MHB is a site-specific environmentally sensitive probe that can provide structural details about amyloid fibrils and their polymorphs.

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