Segmental Isotopic Labeling of a Central Domain in a Multidomain Protein by ProteinTrans-Splicing Using Only One Robust DnaE Intein

2009 ◽  
Vol 121 (33) ◽  
pp. 6244-6247 ◽  
Author(s):  
Alena E. L. Busche ◽  
A. Sesilja Aranko ◽  
Mehdi Talebzadeh-Farooji ◽  
Frank Bernhard ◽  
Volker Dötsch ◽  
...  
Keyword(s):  
Isotopic Labeling ◽  
Central Domain ◽  
Multidomain Protein ◽  
Segmental Isotopic Labeling

Segmental Isotopic Labeling of a Central Domain in a Multidomain Protein by ProteinTrans-Splicing Using Only One Robust DnaE Intein

2009 ◽  
Vol 48 (33) ◽  
pp. 6128-6131 ◽  
Author(s):  
Alena E. L. Busche ◽  
A. Sesilja Aranko ◽  
Mehdi Talebzadeh-Farooji ◽  
Frank Bernhard ◽  
Volker Dötsch ◽  
...  
Keyword(s):  
Isotopic Labeling ◽  
Central Domain ◽  
Multidomain Protein ◽  
Segmental Isotopic Labeling

Segmental Isotopic Labeling of Ubiquitin Chains To Unravel Monomer-Specific Molecular Behavior

2011 ◽  
Vol 123 (47) ◽  
pp. 11406-11410 ◽  
Author(s):  
Carlos A. Castañeda ◽  
Liat Spasser ◽  
Sudhir N. Bavikar ◽  
Ashraf Brik ◽  
David Fushman
Keyword(s):  
Isotopic Labeling ◽  
Molecular Behavior ◽  
Segmental Isotopic Labeling

scholarly journals Segmental isotopic labeling by asparaginyl endopeptidase-mediated protein ligation

2018 ◽  
Vol 71 (4) ◽  
pp. 225-235 ◽  
Author(s):  
Kornelia M. Mikula ◽  
Luisa Krumwiede ◽  
Andreas Plückthun ◽  
Hideo Iwaï
Keyword(s):  
Isotopic Labeling ◽  
Protein Ligation ◽  
Asparaginyl Endopeptidase ◽  
Segmental Isotopic Labeling

scholarly journals Optimization of sortase A ligation for flexible engineering of complex protein systems

2020 ◽  
Vol 295 (9) ◽  
pp. 2664-2675 ◽  
Author(s):  
Jess Li ◽  
Yue Zhang ◽  
Olivier Soubias ◽  
Domarin Khago ◽  
Fa-an Chao ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
High Efficiency ◽  
Isotopic Labeling ◽  
Side Reactions ◽  
Sortase A ◽  
Chemical Ligation ◽  
Multidomain Protein ◽  
Domain Specific ◽  
Wide Range ◽  
Complex Protein

Engineering and bioconjugation of proteins is a critically valuable tool that can facilitate a wide range of biophysical and structural studies. The ability to orthogonally tag or label a domain within a multidomain protein may be complicated by undesirable side reactions to noninvolved domains. Furthermore, the advantages of segmental (or domain-specific) isotopic labeling for NMR, or deuteration for neutron scattering or diffraction, can be realized by an efficient ligation procedure. Common methods—expressed protein ligation, protein trans-splicing, and native chemical ligation—each have specific limitations. Here, we evaluated the use of different variants of Staphylococcus aureus sortase A for a range of ligation reactions and demonstrate that conditions can readily be optimized to yield high efficiency (i.e. completeness of ligation), ease of purification, and functionality in detergents. These properties may enable joining of single domains into multidomain proteins, lipidation to mimic posttranslational modifications, and formation of cyclic proteins to aid in the development of nanodisc membrane mimetics. We anticipate that the method for ligating separate domains into a single functional multidomain protein reported here may enable many applications in structural biology.

scholarly journals Effects of an HIV-1 maturation inhibitor on the structure and dynamics of CA-SP1 junction helices in virus-like particles

2020 ◽  
Vol 117 (19) ◽  
pp. 10286-10293 ◽  
Author(s):  
Sebanti Gupta ◽  
John M. Louis ◽  
Robert Tycko
Keyword(s):  
Chemical Shifts ◽  
Isotopic Labeling ◽  
Relaxation Rates ◽  
Virus Like Particles ◽  
Gag Polyprotein ◽  
Structure And Dynamics ◽  
Maturation Inhibitor ◽  
Rna Maturation ◽  
Segmental Isotopic Labeling ◽  
Hiv 1

HIV-1 maturation involves conversion of the immature Gag polyprotein lattice, which lines the inner surface of the viral membrane, to the mature capsid protein (CA) lattice, which encloses the viral RNA. Maturation inhibitors such as bevirimat (BVM) bind within six-helix bundles, formed by a segment that spans the junction between the CA and spacer peptide 1 (SP1) subunits of Gag, and interfere with cleavage between CA and SP1 catalyzed by the HIV-1 protease (PR). We report solid-state NMR (ssNMR) measurements on spherical virus-like particles (VLPs), facilitated by segmental isotopic labeling, that provide information about effects of BVM on the structure and dynamics of CA–SP1 junction helices in the immature lattice. Although BVM strongly blocks PR-catalyzed CA–SP1 cleavage in VLPs and blocks conversion of VLPs to tubular CA assemblies, 15N and 13C ssNMR chemical shifts of segmentally labeled VLPs with and without BVM are very similar, indicating that interaction with BVM does not alter the six-helix bundle structure appreciably. Only the 15N chemical shift of A280 (the first residue of SP1) changes significantly, consistent with BVM binding to an internal ring of hydrophobic side chains of L279 residues. Measurements of transverse 15N spin relaxation rates reveal a reduction in the amplitudes and/or timescales of backbone N–H bond motions, corresponding to a rigidification of the six-helix bundles. Overall, our data show that inhibition of HIV-1 maturation by BVM involves changes in structure and dynamics that are surprisingly subtle, but still sufficient to produce a large effect on CA–SP1 cleavage.

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