Thermal versus guanidine-induced unfolding of ubiquitin. An analysis in terms of the contributions from charge-charge interactions to protein stability.


Abstract

We have characterized the guanidine-induced unfolding of both yeast and bovine ubiquitin at 25 degrees C and in the acidic pH range on the basis of fluorescence and circular dichroism measurements. Unfolding Gibbs energy changes calculated by linear extrapolation from high guanidine unfolding data are found to depend very weakly on pH. A simple explanation for this result involves the two following assumptions: (1) charged atoms of ionizable groups are exposed to the solvent in native ubiquitin (as supported by accessible surface area calculations), and Gibbs energy contributions associated with charge desolvation upon folding (a source of pK shifts) are small; (2) charge-charge interactions (another source of pK shifts upon folding) are screened out in concentrated guanidinium chloride solutions. We have also characterized the thermal unfolding of both proteins using differential scanning calorimetry. Unfolding Gibbs energy changes calculated from the calorimetric data do depend strongly on pH, a result that we attribute to the pH dependence of charge-charge interactions (not eliminated in the absence of guanidine). In fact, we find good agreement between the difference between the two series of experimental unfolding Gibbs energy changes (determined from high guanidine unfolding data by linear extrapolation and from thermal denaturation data in the absence of guanidine) and the theoretical estimates of the contribution from charge-charge interactions to the Gibbs energy change for ubiquitin unfolding obtained by using the solvent-accessibility-corrected Tanford-Kirkwood model, together with the Bashford-Karplus (reduced-set-of-sites) approximation. This contribution is found to be stabilizing at neutral pH, because most charged groups on the native protein interact mainly with groups of the opposite charge, a fact that, together with the absence of large charge-desolvation contributions, may explain the high stability of ubiquitin at neutral pH. In general, our analysis suggests the possibility of enhancing protein thermal stability by adequately redesigning the distribution of solvent-exposed, charged residues on the native protein surface. Study holds ProTherm entries: 5413, 5414 Extra Details: accessible surface area; charge-charge interactions;,pH dependence; Tanford-Kirkwood model

Submission Details

ID: s2N4wdmy3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:30 p.m.

Version: 1

Publication Details
Ibarra-Molero B;Loladze VV;Makhatadze GI;Sanchez-Ruiz JM,Biochemistry (1999) Thermal versus guanidine-induced unfolding of ubiquitin. An analysis in terms of the contributions from charge-charge interactions to protein stability. PMID:10387059
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
3OLM 2010-08-26T00:00:00+0000 2.5 Structure and Function of a Ubiquitin Binding Site within the Catalytic Domain of a HECT Ubiquitin Ligase
4LCD 2013-06-21T00:00:00+0000 3.1 Structure of an Rsp5xUbxSna3 complex: Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3
5HPL 2016-01-20T00:00:00+0000 2.31 System-wide modulation of HECT E3 ligases with selective ubiquitin variant probes: Rsp5 and UbV R5.4

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 E3 ubiquitin-protein ligase RSP5 P39940 RSP5_YEAST
97.5 B Ubiquitin-binding protein CUE2 P31753 RS27A_ASPOF
100.0 Polyubiquitin-C P84589 UBIQ_LUMTE
90.8 B Ubiquitin-binding protein CUE2 Q39256 UBQ8_ARATH
97.1 B Ubiquitin-binding protein CUE2 P59272 RS27A_DAUCA
94.7 Polyubiquitin-C P69200 RL40_LEITA
95.9 Polyubiquitin-C P14797 RS27A_DICDI
97.2 B Ubiquitin-binding protein CUE2 P27923 RS27A_MAIZE
97.2 B Ubiquitin-binding protein CUE2 P47905 RS27A_LUPAL
97.2 B Ubiquitin-binding protein CUE2 P0CG87 RS272_HORVU
97.2 B Ubiquitin-binding protein CUE2 P0CG86 RS271_HORVU
97.2 B Ubiquitin-binding protein CUE2 P59233 R27AC_ARATH
97.2 B Ubiquitin-binding protein CUE2 P51431 R27AB_ORYSJ
97.2 B Ubiquitin-binding protein CUE2 P59232 R27AB_ARATH
97.2 B Ubiquitin-binding protein CUE2 Q9ARZ9 R27AA_ORYSJ
100.0 B Ubiquitin-binding protein CUE2 P0C8R3 RS27B_SCHPO
100.0 B Ubiquitin-binding protein CUE2 P0C016 RS27A_SCHPO
94.7 Polyubiquitin-C P21899 RL40_TRYBB
97.4 Polyubiquitin-C P69201 RL40_LEIMA
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
94.7 B Ubiquitin-binding protein CUE2 P22589 UBIQP_PHYIN
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
97.4 Polyubiquitin-C P14794 RL40_DICDI
96.1 Polyubiquitin-C P14795 RL40_TRYCR
98.7 Polyubiquitin-C P46575 RL40_EIMBO
96.1 Polyubiquitin-C P0CH27 RL402_TRYCR
97.4 Polyubiquitin-C P0CG82 UBIQP_TETPY
94.7 B Ubiquitin-binding protein CUE2 P23324 UBIQP_EUPEU
94.7 B Ubiquitin-binding protein CUE2 P0C032 RUB3_ORYSJ
97.4 Polyubiquitin-C P0DJ25 RL40_TETTS
97.4 Polyubiquitin-C P33190 RL40_TETPY
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
98.7 B Ubiquitin-binding protein CUE2 P14799 RS27A_NEUCR
96.1 B Ubiquitin-binding protein CUE2 P49633 RL40_ACACA
94.7 B Ubiquitin-binding protein CUE2 Q9FHQ6 UBQ9_ARATH
98.7 Polyubiquitin-C P59669 UBIQP_GEOCY
98.7 Polyubiquitin-C P0CG71 UBIQ1_CAEEL
98.7 Polyubiquitin-C P49632 RL40_CAEEL
97.4 Polyubiquitin-C P0CH10 RL403_CHLRE
97.4 Polyubiquitin-C P0CH11 RL401_CHLRE
96.1 B Ubiquitin-binding protein CUE2 Q3E7K8 UBQ12_ARATH
96.1 B Ubiquitin-binding protein CUE2 P42739 UBIQP_ACEPE
97.4 B Ubiquitin-binding protein CUE2 P42740 UBIQP_AGLNE
100.0 B Ubiquitin-binding protein CUE2 P05759 RS31_YEAST
100.0 B Ubiquitin-binding protein CUE2 P69061 RS27A_KLULA
97.4 B Ubiquitin-binding protein CUE2 P0CH32 UBQ4_ARATH
97.4 B Ubiquitin-binding protein CUE2 Q58G87 UBQ3_ORYSJ
97.4 B Ubiquitin-binding protein CUE2 Q1EC66 UBQ3_ARATH
97.4 B Ubiquitin-binding protein CUE2 Q3E7T8 UBQ14_ARATH
97.4 B Ubiquitin-binding protein CUE2 P0CH33 UBQ11_ARATH
97.4 B Ubiquitin-binding protein CUE2 Q8H159 UBQ10_ARATH
97.4 B Ubiquitin-binding protein CUE2 P69326 UBIQ_WHEAT
97.4 B Ubiquitin-binding protein CUE2 P69317 UBIQ_LUPPO
97.4 B Ubiquitin-binding protein CUE2 P69313 UBIQ_HELAN
97.4 B Ubiquitin-binding protein CUE2 P69310 UBIQ_AVESA
97.4 B Ubiquitin-binding protein CUE2 P69325 UBIQP_SOYBN
97.4 B Ubiquitin-binding protein CUE2 P69322 UBIQP_PEA
97.4 B Ubiquitin-binding protein CUE2 P69315 UBIQP_LINUS
97.4 B Ubiquitin-binding protein CUE2 P0CG83 UBIQP_HORVU
97.4 B Ubiquitin-binding protein CUE2 P69309 UBIQP_AVEFA
97.4 B Ubiquitin-binding protein CUE2 P0CG84 UBI4P_NICSY
97.4 B Ubiquitin-binding protein CUE2 P0CH05 UBI2P_PETCR
97.4 B Ubiquitin-binding protein CUE2 P0CH04 UBI1P_PETCR
97.4 B Ubiquitin-binding protein CUE2 P0CG85 UBI1P_NICSY
97.4 B Ubiquitin-binding protein CUE2 P0C031 RUB2_ORYSJ
97.4 B Ubiquitin-binding protein CUE2 Q8RUC6 RUB2_ARATH
97.4 B Ubiquitin-binding protein CUE2 P0C030 RUB1_ORYSJ
97.4 B Ubiquitin-binding protein CUE2 P0C073 RUB1_DESAN
97.4 B Ubiquitin-binding protein CUE2 Q9SHE7 RUB1_ARATH
97.4 B Ubiquitin-binding protein CUE2 P62981 RS27A_SOLTU
97.4 B Ubiquitin-binding protein CUE2 P62980 RS27A_SOLLC
97.4 B Ubiquitin-binding protein CUE2 P49636 RL40_NICSY
97.4 B Ubiquitin-binding protein CUE2 P51423 RL40_BRARP
97.4 B Ubiquitin-binding protein CUE2 P0CH35 RL40B_ORYSJ
97.4 B Ubiquitin-binding protein CUE2 Q42202 RL40B_ARATH
97.4 B Ubiquitin-binding protein CUE2 P0CH34 RL40A_ORYSJ
97.4 B Ubiquitin-binding protein CUE2 B9DHA6 RL40A_ARATH
97.4 B Ubiquitin-binding protein CUE2 P59271 R27AA_ARATH
98.7 B Ubiquitin-binding protein CUE2 P0CG70 UBI4P_NEUCR
98.7 B Ubiquitin-binding protein CUE2 P0C224 RL40_NEUCR
98.7 B Ubiquitin-binding protein CUE2 P19848 UBIQ_COPCO
100.0 B Ubiquitin-binding protein CUE2 P0CG63 UBI4P_YEAST
100.0 B Ubiquitin-binding protein CUE2 P0CG72 UBI4P_SCHPO
100.0 B Ubiquitin-binding protein CUE2 P0CG75 UBI4P_KLULA
100.0 B Ubiquitin-binding protein CUE2 P0CG74 UBI4P_CANAX
100.0 B Ubiquitin-binding protein CUE2 P0CG73 UBI1P_CANAX
100.0 B Ubiquitin-binding protein CUE2 P40909 RL40_CRYNJ
100.0 B Ubiquitin-binding protein CUE2 P0CH09 RL40B_YEAST
100.0 B Ubiquitin-binding protein CUE2 P0CH08 RL40A_YEAST
100.0 B Ubiquitin-binding protein CUE2 P0CH07 RL402_SCHPO
100.0 B Ubiquitin-binding protein CUE2 P0CH06 RL401_SCHPO
100.0 A Ubiquitin-binding protein CUE2 P36075 CUE2_YEAST