Contribution of the 30/36 hydrophobic contact at the C-terminus of the alpha-helix to the stability of the ubiquitin molecule.


Abstract

The contribution of the hydrophobic contact in the C-capping motif of the alpha-helix to the thermodynamic stability of the ubiquitin molecule has been analyzed. For this, 16 variants of ubiquitin containing the full combinatorial set of four nonpolar residues Val, Ile, Leu, and Phe at C4 (Ile30) and C' ' (Ile36) positions were generated. The secondary structure content as estimated using far-UV circular dichroism (CD) spectroscopy of all but Phe variants at position 30 did not show notable changes upon substitutions. The thermodynamic stability of these ubiquitin variants was measured using differential scanning calorimetry, and it was shown that all variants have lower stability as measured by decreases in the Gibbs energy. Since in some cases the decrease in stability was so dramatic that it rendered an unfolded protein, it was therefore concluded that, despite apparent preservation of the secondary structure, the 30/36 hydrophobic contact is essential for the stability of the ubiquitin molecule. The decrease in the Gibbs energy in many cases was found to be accompanied by a large (up to 25%) decrease in the enthalpy of unfolding, particularly significant in the variants containing Ile to Leu substitutions. This decrease in enthalpy of unfolding is proposed to be primarily the result of the perturbed packing interactions in the native state of the Ile --> Leu variants. The analysis of these data and comparison with effects of similar amino acid substitutions on the stability of other model systems suggest that Ile --> Leu substitutions cannot be isoenergetic at the buried site. Study holds ProTherm entries: 8105, 8106, 8107, 8108, 8109, 8110, 8111, 8112, 8113, 8114, 8115, 8116, 8117, 8118, 8119, 8120, 8121, 8122, 8123, 8124, 8125, 8126, 8127, 8128, 8129, 8130, 8131, 8132, 8133, 8134, 8135, 8136, 8137, 8138, 8139, 8140, 8141, 8142, 8143, 8144, 8145, 8146, 8147, 8148, 8149, 8150, 8151, 8152, 8153, 8154, 8155, 8156, 8157, 8158, 8159, 8160, 8161, 8162, 8163, 8164, 8165, 8166, 8167, 8168, 8169, 8170, 8171, 8172, 8173, 8174, 8175, 8176, 8177, 8178, 8179, 8180, 8181, 8182, 8183, 8184, 8185, 8186, 8187, 8188, 8189, 8190, 8191, 8192, 8193, 8194, 8195, 8196, 8197, 8198, 8199, 14426, 14427, 14428, 14429, 14430, 14431, 14432, 14433, 14434, 14435, 14436, 14437, 14438 Extra Details: hydrophobic contact; nonpolar residues; secondary structure content;,packing interactions

Submission Details

ID: uBteUt3S4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:35 p.m.

Version: 1

Publication Details
Thomas ST;Makhatadze GI,Biochemistry (2000) Contribution of the 30/36 hydrophobic contact at the C-terminus of the alpha-helix to the stability of the ubiquitin molecule. PMID:10956017
Additional Information

Study Summary

Number of data points 295
Proteins E3 ubiquitin-protein ligase RSP5 ; Ubiquitin-binding protein CUE2
Unique complexes 15
Assays/Quantities/Protocols Experimental Assay: dHcal temp:50.0 C, pH:3.0, ionic:-: -, prot_conc:1-50 mg/ml ; Experimental Assay: dCp temp:50.0 C, ionic:-: -, prot_conc:1-50 mg/ml ; Experimental Assay: dG ; Experimental Assay: dCp prot_conc:1,50 mg/ml, ionic:: ; Experimental Assay: dHcal buffers:Sodium acetate: 10 mM, pH:4.0, ionic:: , prot_conc:1,50 mg/ml ; Experimental Assay: Tm buffers:Sodium acetate: 10 mM, pH:4.0 ; Experimental Assay: dHcal pH:3.75, buffers:Sodium acetate: 10 mM, ionic:: , prot_conc:1,50 mg/ml ; Experimental Assay: Tm pH:3.75, buffers:Sodium acetate: 10 mM ; Experimental Assay: dHcal pH:2.25, prot_conc:1,50 mg/ml, ionic:: ; Experimental Assay: Tm pH:2.25, buffers:glycine-HCl: 10 mM ; Experimental Assay: dTm pH:2.5 ; Experimental Assay: dHcal buffers:Sodium acetate: 10 mM, prot_conc:1,50 mg/ml, ionic:: , pH:4.5 ; Experimental Assay: Tm buffers:Sodium acetate: 10 mM, pH:4.5 ; Experimental Assay: dTm pH:3.25 ; Experimental Assay: dTm pH:3.0 ; Experimental Assay: dHcal buffers:Sodium acetate: 10 mM, ionic:: , pH:3.5, prot_conc:1,50 mg/ml ; Experimental Assay: Tm buffers:Sodium acetate: 10 mM, pH:3.5 ; Experimental Assay: dHcal prot_conc:1,50 mg/ml, ionic:: , pH:3.25 ; Experimental Assay: Tm pH:3.25, buffers:glycine-HCl: 10 mM ; Experimental Assay: dHcal prot_conc:1,50 mg/ml, pH:3.0, ionic:: ; Experimental Assay: Tm pH:3.0, buffers:glycine-HCl: 10 mM ; Experimental Assay: dTm pH:2.75 ; Experimental Assay: dHcal prot_conc:1,50 mg/ml, ionic:: , pH:2.75 ; Experimental Assay: Tm pH:2.75, buffers:glycine-HCl: 10 mM ; Experimental Assay: dHcal prot_conc:1,50 mg/ml, pH:2.5, ionic:: ; Experimental Assay: Tm pH:2.5, buffers:glycine-HCl: 10 mM ; Experimental Assay: dHcal prot_conc:1,50 mg/ml, ionic:: , pH:2.0 ; Experimental Assay: Tm pH:2.0, buffers:glycine-HCl: 10 mM ; Derived Quantity: ddG ; Derived Quantity: dTm buffers:Sodium acetate: 10 mM, pH:3.5
Libraries Mutations for sequence A:NDDHESKLSILMDMFPAISKSKLQVHLLENNNDLDLTIGLLLKENDDKS/B:MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG

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
1BT0 1998-09-02T00:00:00+0000 1.7 STRUCTURE OF UBIQUITIN-LIKE PROTEIN, RUB1
1OTR 2003-03-22T00:00:00+0000 0 Solution Structure of a CUE-Ubiquitin Complex
6NYA 2019-02-11T00:00:00+0000 2.06 Crystal Structure of ubiquitin E1 (Uba1) in complex with Ubc3 (Cdc34) and ubiquitin
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
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
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
6PX3 2019-07-24T00:00:00+0000 4.1 Set2 bound to nucleosome
5TTE 2016-11-03T00:00:00+0000 3.5 Crystal Structure of an RBR E3 ubiquitin ligase in complex with an E2-Ub thioester intermediate mimic
7K5J 2020-09-16T00:00:00+0000 3.42 Structure of an E1-E2-ubiquitin thioester mimetic
6PX1 2019-07-24T00:00:00+0000 3.3 Set2 bound to nucleosome

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
95.2 B Ubiquitin-binding protein CUE2 P84589 UBIQ_LUMTE
90.8 B Ubiquitin-binding protein CUE2 Q39256 UBQ8_ARATH
97.1 B Ubiquitin-binding protein CUE2 P59272 RS27A_DAUCA
90.8 B Ubiquitin-binding protein CUE2 P69200 RL40_LEITA
93.2 B Ubiquitin-binding protein CUE2 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
92.1 B Ubiquitin-binding protein CUE2 P21899 RL40_TRYBB
93.4 B Ubiquitin-binding protein CUE2 P69201 RL40_LEIMA
96.0 B Ubiquitin-binding protein CUE2 P62982 RS27A_RAT
96.0 B Ubiquitin-binding protein CUE2 P62983 RS27A_MOUSE
96.0 B Ubiquitin-binding protein CUE2 P68200 RS27A_ICTPU
96.0 B Ubiquitin-binding protein CUE2 P62979 RS27A_HUMAN
96.0 B Ubiquitin-binding protein CUE2 P79781 RS27A_CHICK
96.0 B Ubiquitin-binding protein CUE2 P62978 RS27A_CAVPO
96.0 B Ubiquitin-binding protein CUE2 P62992 RS27A_BOVIN
94.7 B Ubiquitin-binding protein CUE2 Q8SWD4 UBIQ_ENCCU
94.7 B Ubiquitin-binding protein CUE2 P23398 UBIQP_STRPU
94.7 B Ubiquitin-binding protein CUE2 P22589 UBIQP_PHYIN
94.7 B Ubiquitin-binding protein CUE2 P0CG88 UBIQJ_DICDI
94.7 B Ubiquitin-binding protein CUE2 P0CG80 UBIQI_DICDI
94.7 B Ubiquitin-binding protein CUE2 P0CG81 UBIQH_DICDI
94.7 B Ubiquitin-binding protein CUE2 P0CG79 UBIQG_DICDI
94.7 B Ubiquitin-binding protein CUE2 P0CG78 UBIQF_DICDI
94.7 B Ubiquitin-binding protein CUE2 P0CG77 UBIQD_DICDI
94.7 B Ubiquitin-binding protein CUE2 P0CG76 UBIQA_DICDI
94.7 B Ubiquitin-binding protein CUE2 P14794 RL40_DICDI
94.7 B Ubiquitin-binding protein CUE2 P14795 RL40_TRYCR
94.7 B Ubiquitin-binding protein CUE2 P46575 RL40_EIMBO
94.7 B Ubiquitin-binding protein CUE2 P0CH27 RL402_TRYCR
94.7 B Ubiquitin-binding protein CUE2 P0CG82 UBIQP_TETPY
94.7 B Ubiquitin-binding protein CUE2 P23324 UBIQP_EUPEU
94.7 B Ubiquitin-binding protein CUE2 P0C032 RUB3_ORYSJ
94.7 B Ubiquitin-binding protein CUE2 P0DJ25 RL40_TETTS
94.7 B Ubiquitin-binding protein CUE2 P33190 RL40_TETPY
96.1 B Ubiquitin-binding protein CUE2 P62975 UBIQ_RABIT
96.1 B Ubiquitin-binding protein CUE2 P68197 UBIQ_CERCA
96.1 B Ubiquitin-binding protein CUE2 Q865C5 UBIQ_CAMDR
96.1 B Ubiquitin-binding protein CUE2 P62972 UBIQP_XENLA
96.1 B Ubiquitin-binding protein CUE2 P0CG69 UBIQP_DROME
96.1 B Ubiquitin-binding protein CUE2 P62976 UBIQP_CRIGR
96.1 B Ubiquitin-binding protein CUE2 Q63429 UBC_RAT
96.1 B Ubiquitin-binding protein CUE2 P0CG61 UBC_PONPY
96.1 B Ubiquitin-binding protein CUE2 P0CG68 UBC_PIG
96.1 B Ubiquitin-binding protein CUE2 P0CG64 UBC_PANTR
96.1 B Ubiquitin-binding protein CUE2 P0CG50 UBC_MOUSE
96.1 B Ubiquitin-binding protein CUE2 P0CG48 UBC_HUMAN
96.1 B Ubiquitin-binding protein CUE2 P0CG66 UBC_GORGO
96.1 B Ubiquitin-binding protein CUE2 P0CH28 UBC_BOVIN
96.1 B Ubiquitin-binding protein CUE2 P0CG55 UBB_SHEEP
96.1 B Ubiquitin-binding protein CUE2 P0CG51 UBB_RAT
96.1 B Ubiquitin-binding protein CUE2 P0CG60 UBB_PONPY
96.1 B Ubiquitin-binding protein CUE2 P0CG65 UBB_PANTR
96.1 B Ubiquitin-binding protein CUE2 P0CG49 UBB_MOUSE
96.1 B Ubiquitin-binding protein CUE2 P0CG47 UBB_HUMAN
96.1 B Ubiquitin-binding protein CUE2 Q8MKD1 UBB_HORSE
96.1 B Ubiquitin-binding protein CUE2 P0CG67 UBB_GORGO
96.1 B Ubiquitin-binding protein CUE2 P0CG62 UBB_CHICK
96.1 B Ubiquitin-binding protein CUE2 P0CG54 UBB_CAVPO
96.1 B Ubiquitin-binding protein CUE2 P0CG53 UBB_BOVIN
96.1 B Ubiquitin-binding protein CUE2 P68203 RS27A_SPOFR
96.1 B Ubiquitin-binding protein CUE2 P68202 RS27A_PLUXY
96.1 B Ubiquitin-binding protein CUE2 P29504 RS27A_MANSE
96.1 B Ubiquitin-binding protein CUE2 P15357 RS27A_DROME
96.1 B Ubiquitin-binding protein CUE2 P0C276 RL40_SHEEP
96.1 B Ubiquitin-binding protein CUE2 P62986 RL40_RAT
96.1 B Ubiquitin-binding protein CUE2 P0C275 RL40_PONPY
96.1 B Ubiquitin-binding protein CUE2 P63053 RL40_PIG
96.1 B Ubiquitin-binding protein CUE2 P68205 RL40_OPHHA
96.1 B Ubiquitin-binding protein CUE2 P62984 RL40_MOUSE
96.1 B Ubiquitin-binding protein CUE2 P0C273 RL40_MACFA
96.1 B Ubiquitin-binding protein CUE2 P62987 RL40_HUMAN
96.1 B Ubiquitin-binding protein CUE2 P63052 RL40_FELCA
96.1 B Ubiquitin-binding protein CUE2 P18101 RL40_DROME
96.1 B Ubiquitin-binding protein CUE2 P63050 RL40_CANLF
96.1 B Ubiquitin-binding protein CUE2 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
96.1 B Ubiquitin-binding protein CUE2 P59669 UBIQP_GEOCY
96.1 B Ubiquitin-binding protein CUE2 P0CG71 UBIQ1_CAEEL
96.1 B Ubiquitin-binding protein CUE2 P49632 RL40_CAEEL
96.1 B Ubiquitin-binding protein CUE2 P0CH10 RL403_CHLRE
96.1 B Ubiquitin-binding protein CUE2 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