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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 (ΔG f°) 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 (ΔV f) was significantly larger for V67 (71 ± 2 mL mol –1) than for L67 (58 ± 3 mL mol –1) inteins. The measured difference in ΔV f (13 ± 3 mL mol –1) roughly corresponds to the volume of the additional methylene group for Leu, supporting the notion that themore » 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.« 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:
Journal Article: 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. doi: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. doi: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. doi: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 = {Wed May 10 00:00:00 EDT 2017},
month = {Wed May 10 00:00:00 EDT 2017}
}

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  • The mechanism of recA polA lethality in Escherichia coli has been studied. Complementation tests have indicated that both the 5{prime} {yields} 3{prime} exonuclease and the polymerization activities of DNA polymerase I are essential for viability in the absence of RecA protein, whereas the viability and DNA replication of DNA polymerase I-defective cells depend on the recombinase activity of RecA. An alkaline sucrose gradient sedimentation analysis has indicated that RecA has only a minor role in Okazaki fragment processing. Double-strand break repair is proposed for the major role of RecA in the absence of DNA polymerase I. The lexA(Def)::Tn5 mutation hasmore » previously been shown to suppress the temperature-sensitive growth of recA200(Ts) polA25::spc mutants. The lexA(Def) mutation can alleviate impaired DNA synthesis in the recA200(Ts) polA25::spc mutant cells at the restrictive temperature. recF{sup +} is essential for this suppression pathway, recJ and recQ mutations have minor but significant adverse effects on the suppression. The recA200(Ts) allele in the recA200(Ts) polA25::spc lexA(Def) mutant can be replaced by {Delta}recA, indicating that the lexA(Def)-induced suppression is RecA independent. lexA(Def) reduces the sensitivity of {Delta}recA polA25::spc cells to UV damage by {approximately}10{sup 4}-fold. lexA(Def) also restores P1 transduction proficiency to the {Delta}recA polA25::spc mutant to a level that is 7.3% of the recA{sup +} wild type. These results suggest that lexA(Def) activates a RecA-independent, RecF-dependent recombination repair pathway that suppresses the defect in DNA replication in recA polA double mutants. 52 refs., 7 figs., 5 tabs.« less
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  • Light scattering has been used to monitor and distinguish between two types of aggregation reactions observed with the recA protein of Escherichia coli. These are (1) the cooperative binding of recA protein to ssDNA in a pathway leading to DNA strand exchange and (2) the formation of free filaments by recA protein in the absence of DNA. Free filament formation requires Mg/sup 2 +/, is very sensitive to ionic strength, and occurs in the absence of single-stranded DNA and RNA. Turbidity measurements indicate that free recA filaments exhibit properties consistent with rigid rods which are 1 micron or more inmore » length. A kinetically distinct nucleation step in free filament formation is observed under some conditions and becomes rate limiting at high pH. Ninety-degree light scattering was employed to measure binding of recA protein to ssDNA under conditions that either favor or block free filament formation. recA protein saturates ssDNA at a stoichiometric ratio of approximately four nucleotide residues per recA monomer. When free filament formation is blocked by various means, the apparent dissociation constant of the recA X ssDNA complex is approximately 10 nM. Under conditions in which free recA filaments form readily, however, the apparent dissociation constant increases to approximately 1 microM. This dramatic decrease in the observed affinity of recA protein for ssDNA under conditions that permit free filament formation does not reflect a change in the intrinsic affinity of recA protein for ssDNA. Instead, it provides evidence that free filament formation and ssDNA binding by recA protein are competing reactions.« less
  • The authors mapped the human (RECA) and mouse (Reca) homologs of the yeast RAD51 (radiation sensitivity) and Escherichia coli recA genes to human and mouse chromosomes by direct R-banding fluorescence in situ hybridization. This gene was assigned to human chromosome 15q15.1 and to mouse chromosome 2F1, respectively. This is the first report on the precise localization of this gene to human and mouse chromosomes. This gene was mapped to a region on human chromosome 15q15.1 and mouse 2F1 that is believed to be a conserved syntenic group. 16 refs., 1 fig.