skip to main content


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

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
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:
Report Number(s):
Journal ID: ISSN 0006-2960
Grant/Contract Number:
AC52-06NA25396; MCB-1244089; MCB-1517138
Accepted Manuscript
Journal Name:
Additional Journal Information:
Journal Volume: 56; Journal Issue: 21; Journal ID: ISSN 0006-2960
American Chemical Society (ACS)
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)
Country of Publication:
United States
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES; intein; protein splicing; high pressure NMR; protein stability; cavity; volume change of folding
OSTI Identifier: