Contribution of surface salt bridges to protein stability: guidelines for protein engineering.


Abstract

The small globular protein, ubiquitin, contains a pair of oppositely charged residues, K11 and E34, that according to the three-dimensional structure are located on the surface of this protein with a spatial orientation characteristic of a salt bridge. We investigated the strength of this salt bridge and its contribution to the global stability of the ubiquitin molecule. Using the "double mutant cycle" analysis, the strength of the pairwise interactions between K11 and E34 was estimated to be favorable by 3.6kJ/mol. Further, the salt bridge of the reverse orientation, i.e. E11/K34, can be formed and is found to have a strength (3.8kJ/mol) similar to that of the K11/E34 pair. However, the global stability of the K11/E34 variant of ubiquitin is 2.2kJ/mol higher than that of the E11/K34 variant. The difference in the contribution of the opposing salt bridge orientations to the overall stability of the ubiquitin molecule is attributed to the difference in the charge-charge interactions between residues forming the salt bridge and the rest of the ionizable groups in this protein. On the basis of these results, we concluded that surface salt bridges are stabilizing, but their contribution to the overall protein stability is strongly context-dependent, with charge-charge interactions being the largest determinant. Analysis of 16 salt bridges from six different proteins, for which detailed experimental data on energetics have been reported, support the conclusions made from the analysis of the salt bridge in ubiquitin. Implications of these findings for engineering proteins with enhanced thermostability are discussed. Study holds ProTherm entries: 14406, 14407, 14408, 14409, 14410, 14411, 14412, 14413, 14414, 14450 Extra Details: 1L2N was used as PDB code. surface salt bridges; protein stability; protein engineering

Submission Details

ID: hRjmVMwG4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:45 p.m.

Version: 1

Publication Details
Makhatadze GI;Loladze VV;Ermolenko DN;Chen X;Thomas ST,J. Mol. Biol. (2003) Contribution of surface salt bridges to protein stability: guidelines for protein engineering. PMID:12662936
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 UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 E3 ubiquitin-protein ligase RSP5 P39940 RSP5_YEAST
100.0 A Ubiquitin-binding protein CUE2 P36075 CUE2_YEAST
100.0 B Ubiquitin-binding protein CUE2 P0CH06 RL401_SCHPO
100.0 B Ubiquitin-binding protein CUE2 P0CH07 RL402_SCHPO
100.0 B Ubiquitin-binding protein CUE2 P0CH08 RL40A_YEAST
100.0 B Ubiquitin-binding protein CUE2 P0CH09 RL40B_YEAST
100.0 B Ubiquitin-binding protein CUE2 P40909 RL40_CRYNJ
100.0 B Ubiquitin-binding protein CUE2 P0CG73 UBI1P_CANAX
100.0 B Ubiquitin-binding protein CUE2 P0CG74 UBI4P_CANAX
100.0 B Ubiquitin-binding protein CUE2 P0CG75 UBI4P_KLULA
100.0 B Ubiquitin-binding protein CUE2 P0CG72 UBI4P_SCHPO
100.0 B Ubiquitin-binding protein CUE2 P0CG63 UBI4P_YEAST
98.7 B Ubiquitin-binding protein CUE2 P19848 UBIQ_COPCO
98.7 B Ubiquitin-binding protein CUE2 P0C224 RL40_NEUCR
98.7 B Ubiquitin-binding protein CUE2 P0CG70 UBI4P_NEUCR
97.4 B Ubiquitin-binding protein CUE2 P59271 R27AA_ARATH
97.4 B Ubiquitin-binding protein CUE2 B9DHA6 RL40A_ARATH
97.4 B Ubiquitin-binding protein CUE2 P0CH34 RL40A_ORYSJ
97.4 B Ubiquitin-binding protein CUE2 Q42202 RL40B_ARATH
97.4 B Ubiquitin-binding protein CUE2 P0CH35 RL40B_ORYSJ
97.4 B Ubiquitin-binding protein CUE2 P51423 RL40_BRARP
97.4 B Ubiquitin-binding protein CUE2 P49636 RL40_NICSY
97.4 B Ubiquitin-binding protein CUE2 P62980 RS27A_SOLLC
97.4 B Ubiquitin-binding protein CUE2 P62981 RS27A_SOLTU
97.4 B Ubiquitin-binding protein CUE2 Q9SHE7 RUB1_ARATH
97.4 B Ubiquitin-binding protein CUE2 P0C073 RUB1_DESAN
97.4 B Ubiquitin-binding protein CUE2 P0C030 RUB1_ORYSJ
97.4 B Ubiquitin-binding protein CUE2 Q8RUC6 RUB2_ARATH
97.4 B Ubiquitin-binding protein CUE2 P0C031 RUB2_ORYSJ
97.4 B Ubiquitin-binding protein CUE2 P0CG85 UBI1P_NICSY
97.4 B Ubiquitin-binding protein CUE2 P0CH04 UBI1P_PETCR
97.4 B Ubiquitin-binding protein CUE2 P0CH05 UBI2P_PETCR
97.4 B Ubiquitin-binding protein CUE2 P0CG84 UBI4P_NICSY
97.4 B Ubiquitin-binding protein CUE2 P69309 UBIQP_AVEFA
97.4 B Ubiquitin-binding protein CUE2 P0CG83 UBIQP_HORVU
97.4 B Ubiquitin-binding protein CUE2 P69315 UBIQP_LINUS
97.4 B Ubiquitin-binding protein CUE2 P69322 UBIQP_PEA
97.4 B Ubiquitin-binding protein CUE2 P69325 UBIQP_SOYBN
97.4 B Ubiquitin-binding protein CUE2 P69310 UBIQ_AVESA
97.4 B Ubiquitin-binding protein CUE2 P69313 UBIQ_HELAN
97.4 B Ubiquitin-binding protein CUE2 P69317 UBIQ_LUPPO
97.4 B Ubiquitin-binding protein CUE2 P69326 UBIQ_WHEAT
97.4 B Ubiquitin-binding protein CUE2 Q8H159 UBQ10_ARATH
97.4 B Ubiquitin-binding protein CUE2 P0CH33 UBQ11_ARATH
97.4 B Ubiquitin-binding protein CUE2 Q3E7T8 UBQ14_ARATH
97.4 B Ubiquitin-binding protein CUE2 Q1EC66 UBQ3_ARATH
97.4 B Ubiquitin-binding protein CUE2 Q58G87 UBQ3_ORYSJ
97.4 B Ubiquitin-binding protein CUE2 P0CH32 UBQ4_ARATH
100.0 B Ubiquitin-binding protein CUE2 P69061 RS27A_KLULA
100.0 B Ubiquitin-binding protein CUE2 P05759 RS31_YEAST
97.4 B Ubiquitin-binding protein CUE2 P42740 UBIQP_AGLNE
96.1 B Ubiquitin-binding protein CUE2 P42739 UBIQP_ACEPE
96.1 B Ubiquitin-binding protein CUE2 Q3E7K8 UBQ12_ARATH
96.1 B Ubiquitin-binding protein CUE2 P0CH11 RL401_CHLRE
96.1 B Ubiquitin-binding protein CUE2 P0CH10 RL403_CHLRE
96.1 B Ubiquitin-binding protein CUE2 P49632 RL40_CAEEL
96.1 B Ubiquitin-binding protein CUE2 P0CG71 UBIQ1_CAEEL
96.1 B Ubiquitin-binding protein CUE2 P59669 UBIQP_GEOCY
94.7 B Ubiquitin-binding protein CUE2 Q9FHQ6 UBQ9_ARATH
96.1 B Ubiquitin-binding protein CUE2 P49633 RL40_ACACA
98.7 B Ubiquitin-binding protein CUE2 P14799 RS27A_NEUCR
96.1 B Ubiquitin-binding protein CUE2 P63048 RL40_BOVIN
96.1 B Ubiquitin-binding protein CUE2 P63050 RL40_CANLF
96.1 B Ubiquitin-binding protein CUE2 P18101 RL40_DROME
96.1 B Ubiquitin-binding protein CUE2 P63052 RL40_FELCA
96.1 B Ubiquitin-binding protein CUE2 P62987 RL40_HUMAN
96.1 B Ubiquitin-binding protein CUE2 P0C273 RL40_MACFA
96.1 B Ubiquitin-binding protein CUE2 P62984 RL40_MOUSE
96.1 B Ubiquitin-binding protein CUE2 P68205 RL40_OPHHA
96.1 B Ubiquitin-binding protein CUE2 P63053 RL40_PIG
96.1 B Ubiquitin-binding protein CUE2 P0C275 RL40_PONPY
96.1 B Ubiquitin-binding protein CUE2 P62986 RL40_RAT
96.1 B Ubiquitin-binding protein CUE2 P0C276 RL40_SHEEP
96.1 B Ubiquitin-binding protein CUE2 P15357 RS27A_DROME
96.1 B Ubiquitin-binding protein CUE2 P29504 RS27A_MANSE
96.1 B Ubiquitin-binding protein CUE2 P68202 RS27A_PLUXY
96.1 B Ubiquitin-binding protein CUE2 P68203 RS27A_SPOFR
96.1 B Ubiquitin-binding protein CUE2 P0CG53 UBB_BOVIN
96.1 B Ubiquitin-binding protein CUE2 P0CG54 UBB_CAVPO
96.1 B Ubiquitin-binding protein CUE2 P0CG62 UBB_CHICK
96.1 B Ubiquitin-binding protein CUE2 P0CG67 UBB_GORGO
96.1 B Ubiquitin-binding protein CUE2 Q8MKD1 UBB_HORSE
96.1 B Ubiquitin-binding protein CUE2 P0CG47 UBB_HUMAN
96.1 B Ubiquitin-binding protein CUE2 P0CG49 UBB_MOUSE
96.1 B Ubiquitin-binding protein CUE2 P0CG65 UBB_PANTR
96.1 B Ubiquitin-binding protein CUE2 P0CG60 UBB_PONPY
96.1 B Ubiquitin-binding protein CUE2 P0CG51 UBB_RAT
96.1 B Ubiquitin-binding protein CUE2 P0CG55 UBB_SHEEP
96.1 B Ubiquitin-binding protein CUE2 P0CH28 UBC_BOVIN
96.1 B Ubiquitin-binding protein CUE2 P0CG66 UBC_GORGO
96.1 B Ubiquitin-binding protein CUE2 P0CG48 UBC_HUMAN
96.1 B Ubiquitin-binding protein CUE2 P0CG50 UBC_MOUSE
96.1 B Ubiquitin-binding protein CUE2 P0CG64 UBC_PANTR
96.1 B Ubiquitin-binding protein CUE2 P0CG68 UBC_PIG
96.1 B Ubiquitin-binding protein CUE2 P0CG61 UBC_PONPY
96.1 B Ubiquitin-binding protein CUE2 Q63429 UBC_RAT
96.1 B Ubiquitin-binding protein CUE2 P62976 UBIQP_CRIGR
96.1 B Ubiquitin-binding protein CUE2 P0CG69 UBIQP_DROME
96.1 B Ubiquitin-binding protein CUE2 P62972 UBIQP_XENLA
96.1 B Ubiquitin-binding protein CUE2 Q865C5 UBIQ_CAMDR
96.1 B Ubiquitin-binding protein CUE2 P68197 UBIQ_CERCA
96.1 B Ubiquitin-binding protein CUE2 P62975 UBIQ_RABIT
94.7 B Ubiquitin-binding protein CUE2 P33190 RL40_TETPY
94.7 B Ubiquitin-binding protein CUE2 P0DJ25 RL40_TETTS
94.7 B Ubiquitin-binding protein CUE2 P0C032 RUB3_ORYSJ
94.7 B Ubiquitin-binding protein CUE2 P23324 UBIQP_EUPEU
94.7 B Ubiquitin-binding protein CUE2 P0CG82 UBIQP_TETPY
94.7 B Ubiquitin-binding protein CUE2 P0CH27 RL402_TRYCR
94.7 B Ubiquitin-binding protein CUE2 P46575 RL40_EIMBO
94.7 B Ubiquitin-binding protein CUE2 P14795 RL40_TRYCR
94.7 B Ubiquitin-binding protein CUE2 P14794 RL40_DICDI
94.7 B Ubiquitin-binding protein CUE2 P0CG76 UBIQA_DICDI
94.7 B Ubiquitin-binding protein CUE2 P0CG77 UBIQD_DICDI
94.7 B Ubiquitin-binding protein CUE2 P0CG78 UBIQF_DICDI
94.7 B Ubiquitin-binding protein CUE2 P0CG79 UBIQG_DICDI
94.7 B Ubiquitin-binding protein CUE2 P0CG81 UBIQH_DICDI
94.7 B Ubiquitin-binding protein CUE2 P0CG80 UBIQI_DICDI
94.7 B Ubiquitin-binding protein CUE2 P0CG88 UBIQJ_DICDI
94.7 B Ubiquitin-binding protein CUE2 P22589 UBIQP_PHYIN
94.7 B Ubiquitin-binding protein CUE2 P23398 UBIQP_STRPU
94.7 B Ubiquitin-binding protein CUE2 Q8SWD4 UBIQ_ENCCU
96.0 B Ubiquitin-binding protein CUE2 P62992 RS27A_BOVIN
96.0 B Ubiquitin-binding protein CUE2 P62978 RS27A_CAVPO
96.0 B Ubiquitin-binding protein CUE2 P79781 RS27A_CHICK
96.0 B Ubiquitin-binding protein CUE2 P62979 RS27A_HUMAN
96.0 B Ubiquitin-binding protein CUE2 P68200 RS27A_ICTPU
96.0 B Ubiquitin-binding protein CUE2 P62983 RS27A_MOUSE
96.0 B Ubiquitin-binding protein CUE2 P62982 RS27A_RAT
93.4 B Ubiquitin-binding protein CUE2 P69201 RL40_LEIMA
92.1 B Ubiquitin-binding protein CUE2 P21899 RL40_TRYBB
100.0 B Ubiquitin-binding protein CUE2 P0C016 RS27A_SCHPO
100.0 B Ubiquitin-binding protein CUE2 P0C8R3 RS27B_SCHPO
97.2 B Ubiquitin-binding protein CUE2 Q9ARZ9 R27AA_ORYSJ
97.2 B Ubiquitin-binding protein CUE2 P59232 R27AB_ARATH
97.2 B Ubiquitin-binding protein CUE2 P51431 R27AB_ORYSJ
97.2 B Ubiquitin-binding protein CUE2 P59233 R27AC_ARATH
97.2 B Ubiquitin-binding protein CUE2 P0CG86 RS271_HORVU
97.2 B Ubiquitin-binding protein CUE2 P0CG87 RS272_HORVU
97.2 B Ubiquitin-binding protein CUE2 P47905 RS27A_LUPAL
97.2 B Ubiquitin-binding protein CUE2 P27923 RS27A_MAIZE
93.2 B Ubiquitin-binding protein CUE2 P14797 RS27A_DICDI
90.8 B Ubiquitin-binding protein CUE2 P69200 RL40_LEITA
97.1 B Ubiquitin-binding protein CUE2 P59272 RS27A_DAUCA
90.8 B Ubiquitin-binding protein CUE2 Q39256 UBQ8_ARATH
95.2 B Ubiquitin-binding protein CUE2 P84589 UBIQ_LUMTE
97.5 B Ubiquitin-binding protein CUE2 P31753 RS27A_ASPOF