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Title: V67L Mutation Fills an Internal Cavity To Stabilize RecA Mtu Intein

Abstract

Inteins mediate protein splicing, which has found extensive applications in protein science and biotechnology. In the Mycobacterium tuberculosis RecA mini–mini intein (ΔΔIhh), a single valine to leucine substitution at position 67 (V67L) dramatically increases intein stability and activity. However, crystal structures show that the V67L mutation causes minimal structural rearrangements, with a root-mean-square deviation of 0.2 Å between ΔΔIhh-V67 and ΔΔIhh-L67. Thus, the structural mechanisms for V67L stabilization and activation remain poorly understood. In this paper, we used intrinsic tryptophan fluorescence, high-pressure nuclear magnetic resonance (NMR), and molecular dynamics (MD) simulations to probe the structural basis of V67L stabilization of the intein fold. Guanidine hydrochloride denaturation monitored by fluorescence yielded free energy changes (ΔGf°) of -4.4 and -6.9 kcal mol–1 for ΔΔIhh-V67 and ΔΔIhh-L67, respectively. High-pressure NMR showed that ΔΔIhh-L67 is more resistant to pressure-induced unfolding than ΔΔIhh-V67 is. The change in the volume of folding (ΔVf) was significantly larger for V67 (71 ± 2 mL mol–1) than for L67 (58 ± 3 mL mol–1) inteins. The measured difference in ΔVf (13 ± 3 mL mol–1) roughly corresponds to the volume of the additional methylene group for Leu, supporting the notion that the V67L mutation fills a nearby cavity tomore » enhance intein stability. In addition, we performed MD simulations to show that V67L decreases side chain dynamics and conformational entropy at the active site. Finally, it is plausible that changes in cavities in V67L can also mediate allosteric effects to change active site dynamics and enhance intein activity.« less

Authors:
ORCiD logo [1];  [1];  [2];  [1];  [1];  [3]; ORCiD logo [1]; ORCiD logo [4];  [1]
  1. Rensselaer Polytechnic Inst., Troy, NY (United States). Dept. of Biological Sciences
  2. Aberystwyth Univ., Ceredigion, Wales (United Kingdom). Dept. of Computer Science
  3. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
  4. College of the Holy Cross, Worcester, MA (United States). Dept. of Chemistry
Publication Date:
Research Org.:
Rensselaer Polytechnic Inst., Troy, NY (United States); College of the Holy Cross, Worcester, MA (United States); Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Sponsoring Org.:
USDOE; National Science Foundation (NSF)
OSTI Identifier:
1409771
Report Number(s):
LA-UR-17-23607
Journal ID: ISSN 0006-2960
Grant/Contract Number:  
AC52-06NA25396; MCB-1244089; MCB-1517138
Resource Type:
Accepted Manuscript
Journal Name:
Biochemistry
Additional Journal Information:
Journal Volume: 56; Journal Issue: 21; Journal ID: ISSN 0006-2960
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES; intein; protein splicing; high pressure NMR; protein stability; cavity; volume change of folding

Citation Formats

Zwarycz, Allison S., Fossat, Martin, Akanyeti, Otar, Lin, Zhongqian, Rosenman, David J., Garcia, Angel E., Royer, Catherine A., Mills, Kenneth V., and Wang, Chunyu. V67L Mutation Fills an Internal Cavity To Stabilize RecA Mtu Intein. United States: N. p., 2017. Web. https://doi.org/10.1021/acs.biochem.6b01264.
Zwarycz, Allison S., Fossat, Martin, Akanyeti, Otar, Lin, Zhongqian, Rosenman, David J., Garcia, Angel E., Royer, Catherine A., Mills, Kenneth V., & Wang, Chunyu. V67L Mutation Fills an Internal Cavity To Stabilize RecA Mtu Intein. United States. https://doi.org/10.1021/acs.biochem.6b01264
Zwarycz, Allison S., Fossat, Martin, Akanyeti, Otar, Lin, Zhongqian, Rosenman, David J., Garcia, Angel E., Royer, Catherine A., Mills, Kenneth V., and Wang, Chunyu. Wed . "V67L Mutation Fills an Internal Cavity To Stabilize RecA Mtu Intein". United States. https://doi.org/10.1021/acs.biochem.6b01264. https://www.osti.gov/servlets/purl/1409771.
@article{osti_1409771,
title = {V67L Mutation Fills an Internal Cavity To Stabilize RecA Mtu Intein},
author = {Zwarycz, Allison S. and Fossat, Martin and Akanyeti, Otar and Lin, Zhongqian and Rosenman, David J. and Garcia, Angel E. and Royer, Catherine A. and Mills, Kenneth V. and Wang, Chunyu},
abstractNote = {Inteins mediate protein splicing, which has found extensive applications in protein science and biotechnology. In the Mycobacterium tuberculosis RecA mini–mini intein (ΔΔIhh), a single valine to leucine substitution at position 67 (V67L) dramatically increases intein stability and activity. However, crystal structures show that the V67L mutation causes minimal structural rearrangements, with a root-mean-square deviation of 0.2 Å between ΔΔIhh-V67 and ΔΔIhh-L67. Thus, the structural mechanisms for V67L stabilization and activation remain poorly understood. In this paper, we used intrinsic tryptophan fluorescence, high-pressure nuclear magnetic resonance (NMR), and molecular dynamics (MD) simulations to probe the structural basis of V67L stabilization of the intein fold. Guanidine hydrochloride denaturation monitored by fluorescence yielded free energy changes (ΔGf°) of -4.4 and -6.9 kcal mol–1 for ΔΔIhh-V67 and ΔΔIhh-L67, respectively. High-pressure NMR showed that ΔΔIhh-L67 is more resistant to pressure-induced unfolding than ΔΔIhh-V67 is. The change in the volume of folding (ΔVf) was significantly larger for V67 (71 ± 2 mL mol–1) than for L67 (58 ± 3 mL mol–1) inteins. The measured difference in ΔVf (13 ± 3 mL mol–1) roughly corresponds to the volume of the additional methylene group for Leu, supporting the notion that the V67L mutation fills a nearby cavity to enhance intein stability. In addition, we performed MD simulations to show that V67L decreases side chain dynamics and conformational entropy at the active site. Finally, it is plausible that changes in cavities in V67L can also mediate allosteric effects to change active site dynamics and enhance intein activity.},
doi = {10.1021/acs.biochem.6b01264},
journal = {Biochemistry},
number = 21,
volume = 56,
place = {United States},
year = {2017},
month = {5}
}

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