Engineering a thermostable protein via optimization of charge-charge interactions on the protein surface.


Abstract

A simple theoretical model for increasing the protein stability by adequately redesigning the distribution of charged residues on the surface of the native protein was tested experimentally. Using the molecule of ubiquitin as a model system, we predicted possible amino acid substitutions on the surface of this protein which would lead to an increase in its stability. Experimental validation for this prediction was achieved by measuring the stabilities of single-site-substituted ubiquitin variants using urea-induced unfolding monitored by far-UV CD spectroscopy. We show that the generated variants of ubiquitin are indeed more stable than the wild-type protein, in qualitative agreement with the theoretical prediction. As a positive control, theoretical predictions for destabilizing amino acid substitutions on the surface of the ubiquitin molecule were considered as well. These predictions were also tested experimentally using correspondingly designed variants of ubiquitin. We found that these variants are less stable than the wild-type protein, again in agreement with the theoretical prediction. These observations provide guidelines for rational design of more stable proteins and suggest a possible mechanism of structural stability of proteins from thermophilic organisms. Study holds ProTherm entries: 5978, 5979, 5980, 5981, 5982, 5983, 5984, 5985, 5986, 5987 Extra Details: theoretical model; surface; structural stability;,thermophilic organism

Submission Details

ID: HCqxAkcP4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:31 p.m.

Version: 1

Publication Details
Loladze VV;Ibarra-Molero B;Sanchez-Ruiz JM;Makhatadze GI,Biochemistry (1999) Engineering a thermostable protein via optimization of charge-charge interactions on the protein surface. PMID:10600102
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
5L9U 2016-06-11T00:00:00+0000 6.4 Model of human Anaphase-promoting complex/Cyclosome (APC/C-CDH1) with a cross linked Ubiquitin variant-substrate-UBE2C (UBCH10) complex representing key features of multiubiquitination
5OHM 2017-07-17T00:00:00+0000 3.8 K33-specific affimer bound to K33 diUb
6ZVK 2020-07-24T00:00:00+0000 3.49 The Halastavi arva virus (HalV) intergenic region IRES promotes translation by the simplest possible initiation mechanism
2K39 2008-04-25T00:00:00+0000 0 Recognition dynamics up to microseconds revealed from RDC derived ubiquitin ensemble in solution
2KDE 2009-01-06T00:00:00+0000 0 NMR structure of major S5a (196-306):K48 linked diubiquitin species
3AI5 2010-05-10T00:00:00+0000 1.4 Crystal structure of yeast enhanced green fluorescent protein-ubiquitin fusion protein
6JWI 2019-04-20T00:00:00+0000 2.55 Yeast Npl4 in complex with Lys48-linked diubiquitin
1E0Q 2000-04-05T00:00:00+0000 0 Mutant Peptide from the first N-terminal 17 amino-acid of Ubiquitin
3J7O 2014-08-01T00:00:00+0000 3.5 Structure of the mammalian 60S ribosomal subunit
3J7P 2014-08-01T00:00:00+0000 3.5 Structure of the 80S mammalian ribosome bound to eEF2

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
97.5 Polyubiquitin-C P31753 RS27A_ASPOF
100.0 Polyubiquitin-C P84589 UBIQ_LUMTE
95.7 Polyubiquitin-C P59272 RS27A_DAUCA
95.8 Polyubiquitin-C P0C8R3 RS27B_SCHPO
95.8 Polyubiquitin-C P0C016 RS27A_SCHPO
95.8 Polyubiquitin-C P27923 RS27A_MAIZE
95.8 Polyubiquitin-C P47905 RS27A_LUPAL
95.8 Polyubiquitin-C P0CG87 RS272_HORVU
95.8 Polyubiquitin-C P0CG86 RS271_HORVU
95.8 Polyubiquitin-C P59233 R27AC_ARATH
95.8 Polyubiquitin-C P51431 R27AB_ORYSJ
95.8 Polyubiquitin-C P59232 R27AB_ARATH
95.8 Polyubiquitin-C Q9ARZ9 R27AA_ORYSJ
95.9 Polyubiquitin-C P14797 RS27A_DICDI
96.0 Polyubiquitin-C P14799 RS27A_NEUCR
94.7 Polyubiquitin-C P69200 RL40_LEITA
93.4 Polyubiquitin-C P23324 UBIQP_EUPEU
96.0 Polyubiquitin-C P05759 RS31_YEAST
96.0 Polyubiquitin-C P69061 RS27A_KLULA
94.7 Polyubiquitin-C P21899 RL40_TRYBB
93.4 Polyubiquitin-C Q9FHQ6 UBQ9_ARATH
94.7 Polyubiquitin-C Q3E7K8 UBQ12_ARATH
96.1 Polyubiquitin-C P0CG70 UBI4P_NEUCR
96.1 Polyubiquitin-C P14795 RL40_TRYCR
96.1 Polyubiquitin-C P0C224 RL40_NEUCR
96.1 Polyubiquitin-C P49633 RL40_ACACA
96.1 Polyubiquitin-C P0CH27 RL402_TRYCR
94.7 Polyubiquitin-C P22589 UBIQP_PHYIN
94.7 Polyubiquitin-C P42739 UBIQP_ACEPE
94.7 Polyubiquitin-C P0C032 RUB3_ORYSJ
96.1 Polyubiquitin-C P0CH32 UBQ4_ARATH
96.1 Polyubiquitin-C Q58G87 UBQ3_ORYSJ
96.1 Polyubiquitin-C Q1EC66 UBQ3_ARATH
96.1 Polyubiquitin-C Q3E7T8 UBQ14_ARATH
96.1 Polyubiquitin-C P0CH33 UBQ11_ARATH
96.1 Polyubiquitin-C Q8H159 UBQ10_ARATH
96.1 Polyubiquitin-C P69326 UBIQ_WHEAT
96.1 Polyubiquitin-C P69317 UBIQ_LUPPO
96.1 Polyubiquitin-C P69313 UBIQ_HELAN
96.1 Polyubiquitin-C P69310 UBIQ_AVESA
96.1 Polyubiquitin-C P69325 UBIQP_SOYBN
96.1 Polyubiquitin-C P69322 UBIQP_PEA
96.1 Polyubiquitin-C P69315 UBIQP_LINUS
96.1 Polyubiquitin-C P0CG83 UBIQP_HORVU
96.1 Polyubiquitin-C P69309 UBIQP_AVEFA
97.4 Polyubiquitin-C P0CG88 UBIQJ_DICDI
97.4 Polyubiquitin-C P0CG80 UBIQI_DICDI
97.4 Polyubiquitin-C P0CG81 UBIQH_DICDI
97.4 Polyubiquitin-C P0CG79 UBIQG_DICDI
97.4 Polyubiquitin-C P0CG78 UBIQF_DICDI
97.4 Polyubiquitin-C P0CG77 UBIQD_DICDI
97.4 Polyubiquitin-C P0CG76 UBIQA_DICDI
96.1 Polyubiquitin-C P0CG63 UBI4P_YEAST
96.1 Polyubiquitin-C P0CG72 UBI4P_SCHPO
96.1 Polyubiquitin-C P0CG84 UBI4P_NICSY
96.1 Polyubiquitin-C P0CG75 UBI4P_KLULA
96.1 Polyubiquitin-C P0CG74 UBI4P_CANAX
96.1 Polyubiquitin-C P0CH05 UBI2P_PETCR
96.1 Polyubiquitin-C P0CH04 UBI1P_PETCR
96.1 Polyubiquitin-C P0CG85 UBI1P_NICSY
96.1 Polyubiquitin-C P0CG73 UBI1P_CANAX
96.1 Polyubiquitin-C P0C031 RUB2_ORYSJ
96.1 Polyubiquitin-C Q8RUC6 RUB2_ARATH
96.1 Polyubiquitin-C P0C030 RUB1_ORYSJ
96.1 Polyubiquitin-C P0C073 RUB1_DESAN
96.1 Polyubiquitin-C Q9SHE7 RUB1_ARATH
96.1 Polyubiquitin-C P62981 RS27A_SOLTU
96.1 Polyubiquitin-C P62980 RS27A_SOLLC
96.1 Polyubiquitin-C P49636 RL40_NICSY
97.4 Polyubiquitin-C P14794 RL40_DICDI
96.1 Polyubiquitin-C P40909 RL40_CRYNJ
96.1 Polyubiquitin-C P51423 RL40_BRARP
96.1 Polyubiquitin-C P0CH09 RL40B_YEAST
96.1 Polyubiquitin-C P0CH35 RL40B_ORYSJ
96.1 Polyubiquitin-C Q42202 RL40B_ARATH
96.1 Polyubiquitin-C P0CH08 RL40A_YEAST
96.1 Polyubiquitin-C P0CH34 RL40A_ORYSJ
96.1 Polyubiquitin-C B9DHA6 RL40A_ARATH
96.1 Polyubiquitin-C P0CH07 RL402_SCHPO
96.1 Polyubiquitin-C P0CH06 RL401_SCHPO
96.1 Polyubiquitin-C P59271 R27AA_ARATH
97.4 Polyubiquitin-C P42740 UBIQP_AGLNE
97.4 Polyubiquitin-C P19848 UBIQ_COPCO
97.4 Polyubiquitin-C P0CG82 UBIQP_TETPY
97.4 Polyubiquitin-C P0DJ25 RL40_TETTS
97.4 Polyubiquitin-C P33190 RL40_TETPY
97.4 Polyubiquitin-C P0CH10 RL403_CHLRE
97.4 Polyubiquitin-C P0CH11 RL401_CHLRE
97.4 Polyubiquitin-C P69201 RL40_LEIMA
98.7 Polyubiquitin-C P59669 UBIQP_GEOCY
98.7 Polyubiquitin-C P0CG71 UBIQ1_CAEEL
98.7 Polyubiquitin-C P49632 RL40_CAEEL
100.0 Polyubiquitin-C P62982 RS27A_RAT
100.0 Polyubiquitin-C P62983 RS27A_MOUSE
100.0 Polyubiquitin-C P68200 RS27A_ICTPU
100.0 Polyubiquitin-C P62979 RS27A_HUMAN
100.0 Polyubiquitin-C P79781 RS27A_CHICK
100.0 Polyubiquitin-C P62978 RS27A_CAVPO
100.0 Polyubiquitin-C P62992 RS27A_BOVIN
98.7 Polyubiquitin-C Q8SWD4 UBIQ_ENCCU
98.7 Polyubiquitin-C P23398 UBIQP_STRPU
98.7 Polyubiquitin-C P46575 RL40_EIMBO
100.0 Polyubiquitin-C P62975 UBIQ_RABIT
100.0 Polyubiquitin-C P68197 UBIQ_CERCA
100.0 Polyubiquitin-C Q865C5 UBIQ_CAMDR
100.0 Polyubiquitin-C P62972 UBIQP_XENLA
100.0 Polyubiquitin-C P0CG69 UBIQP_DROME
100.0 Polyubiquitin-C P62976 UBIQP_CRIGR
100.0 Polyubiquitin-C Q63429 UBC_RAT
100.0 Polyubiquitin-C P0CG61 UBC_PONPY
100.0 Polyubiquitin-C P0CG68 UBC_PIG
100.0 Polyubiquitin-C P0CG64 UBC_PANTR
100.0 Polyubiquitin-C P0CG50 UBC_MOUSE
100.0 Polyubiquitin-C P0CG48 UBC_HUMAN
100.0 Polyubiquitin-C P0CG66 UBC_GORGO
100.0 Polyubiquitin-C P0CH28 UBC_BOVIN
100.0 Polyubiquitin-C P0CG55 UBB_SHEEP
100.0 Polyubiquitin-C P0CG51 UBB_RAT
100.0 Polyubiquitin-C P0CG60 UBB_PONPY
100.0 Polyubiquitin-C P0CG65 UBB_PANTR
100.0 Polyubiquitin-C P0CG49 UBB_MOUSE
100.0 Polyubiquitin-C P0CG47 UBB_HUMAN
100.0 Polyubiquitin-C Q8MKD1 UBB_HORSE
100.0 Polyubiquitin-C P0CG67 UBB_GORGO
100.0 Polyubiquitin-C P0CG62 UBB_CHICK
100.0 Polyubiquitin-C P0CG54 UBB_CAVPO
100.0 Polyubiquitin-C P0CG53 UBB_BOVIN
100.0 Polyubiquitin-C P68203 RS27A_SPOFR
100.0 Polyubiquitin-C P68202 RS27A_PLUXY
100.0 Polyubiquitin-C P29504 RS27A_MANSE
100.0 Polyubiquitin-C P15357 RS27A_DROME
100.0 Polyubiquitin-C P0C276 RL40_SHEEP
100.0 Polyubiquitin-C P62986 RL40_RAT
100.0 Polyubiquitin-C P0C275 RL40_PONPY
100.0 Polyubiquitin-C P63053 RL40_PIG
100.0 Polyubiquitin-C P68205 RL40_OPHHA
100.0 Polyubiquitin-C P62984 RL40_MOUSE
100.0 Polyubiquitin-C P0C273 RL40_MACFA
100.0 Polyubiquitin-C P62987 RL40_HUMAN
100.0 Polyubiquitin-C P63052 RL40_FELCA
100.0 Polyubiquitin-C P18101 RL40_DROME
100.0 Polyubiquitin-C P63050 RL40_CANLF
100.0 Polyubiquitin-C P63048 RL40_BOVIN