Charges in the hydrophobic interior of proteins.


Abstract

Charges are inherently incompatible with hydrophobic environments. Presumably for this reason, ionizable residues are usually excluded from the hydrophobic interior of proteins and are found instead at the surface, where they can interact with bulk water. Paradoxically, ionizable groups buried in the hydrophobic interior of proteins play essential roles, especially in biological energy transduction. To examine the unusual properties of internal ionizable groups we measured the pK(a) of glutamic acid residues at 25 internal positions in a stable form of staphylococcal nuclease. Two of 25 Glu residues titrated with normal pK(a) near 4.5; the other 23 titrated with elevated pK(a) values ranging from 5.2-9.4, with an average value of 7.7. Trp fluorescence and far-UV circular dichroism were used to monitor the effects of internal charges on conformation. These data demonstrate that although charges buried in proteins are indeed destabilizing, charged side chains can be buried readily in the hydrophobic core of stable proteins without the need for specialized structural adaptations to stabilize them, and without inducing any major conformational reorganization. The apparent dielectric effect experienced by the internal charges is considerably higher than the low dielectric constants of hydrophobic matter used to represent the protein interior in electrostatic continuum models of proteins. The high thermodynamic stability required for proteins to withstand the presence of buried charges suggests a pathway for the evolution of enzymes, and it underscores the need to mind thermodynamic stability in any strategy for engineering novel or altered enzymatic active sites in proteins. Study holds ProTherm entries: 25857, 25858, 25859, 25860, 25861, 25862, 25863, 25864, 25865, 25866, 25867, 25868, 25869, 25870, 25871, 25872, 25873, 25874, 25875, 25876, 25877, 25878, 25879, 25880, 25881, 25882, 25883, 25884, 25885, 25886, 25887, 25888, 25889, 25890, 25891, 25892, 25893, 25894, 25895, 25896, 25897, 25898, 25899, 25900, 25901, 25902, 25903, 25904, 25905, 25906, 25907, 25908, 25909, 25910, 25911, 25912, 25913, 25914, 25915, 25916, 25917, 25918, 25919, 25920, 25921, 25922, 25923, 25924, 25925, 25926, 25927, 25928, 25929, 25930, 25931, 25932, 25933, 25934, 25935, 25936, 25937, 25938, 25939, 25940, 25941, 25942, 25943, 25944, 25945, 25946, 25947, 25948, 25949, 25950, 25951, 25952, 25953, 25954, 25955, 25956, 25957, 25958, 25959, 25960, 25961, 25962, 25963, 25964, 25965, 25966, 25967, 25968, 25969, 25970, 25971, 25972, 25973, 25974, 25975, 25976, 25977, 25978 Extra Details: dielectric effect; electrostatics; hydration; pKa; bioenergetics

Submission Details

ID: mMUR8RZq3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:56 p.m.

Version: 1

Publication Details
Isom DG;Castañeda CA;Cannon BR;Velu PD;García-Moreno E B,Proc. Natl. Acad. Sci. U.S.A. (2010) Charges in the hydrophobic interior of proteins. PMID:20798341
Additional Information

Study Summary

Number of data points 488
Proteins Thermonuclease ; Thermonuclease
Unique complexes 24
Assays/Quantities/Protocols Experimental Assay: Cm pH:5.5 ; Experimental Assay: m pH:5.5 ; Experimental Assay: dG_H2O pH:5.5 ; Experimental Assay: Cm pH:5.9 ; Experimental Assay: m pH:5.9 ; Experimental Assay: dG_H2O pH:5.9 ; Experimental Assay: Cm pH:4.5 ; Experimental Assay: m pH:4.5 ; Experimental Assay: dG_H2O pH:4.5 ; Experimental Assay: Cm pH:9.9 ; Experimental Assay: m pH:9.9 ; Experimental Assay: dG_H2O pH:9.9 ; Experimental Assay: Cm pH:4.4 ; Experimental Assay: m pH:4.4 ; Experimental Assay: dG_H2O pH:4.4 ; Experimental Assay: Cm pH:9.0 ; Experimental Assay: m pH:9.0 ; Experimental Assay: dG_H2O pH:9.0 ; Experimental Assay: Cm pH:8.0 ; Experimental Assay: m pH:8.0 ; Experimental Assay: dG_H2O pH:8.0 ; Experimental Assay: Cm pH:5.0 ; Experimental Assay: m pH:5.0 ; Experimental Assay: dG_H2O pH:5.0 ; Experimental Assay: Cm pH:9.1 ; Experimental Assay: m pH:9.1 ; Experimental Assay: dG_H2O pH:9.1 ; Experimental Assay: Cm pH:7.9 ; Experimental Assay: m pH:7.9 ; Experimental Assay: dG_H2O pH:7.9 ; Experimental Assay: Cm pH:6.0 ; Experimental Assay: m pH:6.0 ; Experimental Assay: dG_H2O pH:6.0 ; Experimental Assay: Cm pH:4.9 ; Experimental Assay: m pH:4.9 ; Experimental Assay: dG_H2O pH:4.9 ; Experimental Assay: Cm pH:3.9 ; Experimental Assay: m pH:3.9 ; Experimental Assay: dG_H2O pH:3.9 ; Derived Quantity: ddG_H2O pH:5.5 ; Derived Quantity: ddG_H2O pH:5.9 ; Derived Quantity: ddG_H2O pH:4.5 ; Derived Quantity: ddG_H2O pH:9.9 ; Derived Quantity: ddG_H2O pH:4.4 ; Derived Quantity: ddG_H2O pH:9.0 ; Derived Quantity: ddG_H2O pH:8.0 ; Derived Quantity: ddG_H2O pH:5.0 ; Derived Quantity: ddG_H2O pH:9.1 ; Derived Quantity: ddG_H2O pH:7.9 ; Derived Quantity: ddG_H2O pH:6.0 ; Derived Quantity: ddG_H2O pH:4.9 ; Derived Quantity: ddG_H2O pH:3.9
Libraries Mutations for sequence ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETKHPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALVRQGLAKVAYVYKPNNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQ

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
4WRD 2014-10-23T00:00:00+0000 1.65 Crystal structure of Staphylcoccal nulease variant Delta+PHS V66E L125E at cryogenic temperature
2LKV 2011-10-21T00:00:00+0000 0 Staphylococcal Nuclease PHS variant
2M00 2012-10-14T00:00:00+0000 0 Solution structure of staphylococcal nuclease E43S mutant in the presence of ssDNA and Cd2+
2OXP 2007-02-20T00:00:00+0000 2.0 Crystal Structure of Staphylococcal Nuclease mutant V66D/P117G/H124L/S128A
3D4W 2008-05-15T00:00:00+0000 1.9 Crystal structure of Staphylococcal nuclease variant Delta+PHS A109R at cryogenic temperature
3D8G 2008-05-23T00:00:00+0000 1.99 Crystal structure of Staphylococcal nuclease variant Delta+PHS I72R at cryogenic temperature
3MVV 2010-05-04T00:00:00+0000 1.72 Crystal structure of Staphylococcal nuclease variant Delta+PHS F34A at cryogenic temperature
3QOJ 2011-02-10T00:00:00+0000 1.6 Cryogenic structure of Staphylococcal nuclease variant D+PHS/V23K
3QOL 2011-02-10T00:00:00+0000 1.9 Crystal structure of Staphylococcal nuclease variant D+PHS/V23E at pH 6 determined at 100 K
3R3O 2011-03-16T00:00:00+0000 1.9 Crystal structure of Staphylococcal nuclease variant Delta+PHS T62A at cryogenic temperature and with high redundancy

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.3 Thermonuclease Q6GIK1 NUC_STAAR
99.3 Thermonuclease Q8NXI6 NUC_STAAW
99.3 Thermonuclease Q6GB41 NUC_STAAS
99.1 Thermonuclease Q7A6P2 NUC_STAAN
99.1 Thermonuclease Q99VJ0 NUC_STAAM
99.3 Thermonuclease Q5HHM4 NUC_STAAC
100.0 Thermonuclease P00644 NUC_STAAU