Thermodynamic stability of annexin V E17G: equilibrium parameters from an irreversible unfolding reaction.


Abstract

Conformational stability of the membrane-binding protein annexin V E17G has been determined by high-sensitivity differential scanning microcalorimetry (DSC) measurements and by isothermal, guanidinium hydrochloride (GdnHCl)-induced unfolding studies. Wild-type annexin V and the E17G mutant protein studied here are structurally almost identical. Therefore, it can be expected that the present results will not deviate significantly from the stability data of the wild-type molecule. Thermal unfolding is irreversible, while GdnHCl unfolding shows a high degree of reversibility. We were able to demonstrate that characteristic features of annexin V E17G unfolding permit us to extract from the kinetically controlled heat capacity curves thermodynamic equilibrium parameters at the high heating rates. The thermodynamic quantities obtained from the DSC studies in phosphate buffer at pH 7.0 are as follows: t1/2 = 54.7 degrees C (heating rate of 2.34 K min-1), delta H0 = 690 kJ mol-1, and delta Cp = 10.3 kJ mol-1 K-1 which correspondends to a value of delta G0D (20 degrees C) of 53.4 kJ mol-1. When compared on a per gram basis, these thermodynamic parameters classify annexin V E17G as a marginally stable protein. This conclusion is consistent with structural and functional features of the protein that require conformational adaptability for hinge-bending motions and pore formation on interaction with membranes. We observed a large difference between the change in the Gibbs energy value derived from the heat capacity studies and that determined from the GdnHCl unfolding curve. The difference appears to stem from a specific interaction of the protein with the denaturant that results in both a low half-denaturation concentration C1/2 of 1.74 M and a small slope (6.0 kJ L mol-2) of the delta Gapp versus [GdnHCl] plot. The extraordinary interaction of annexin V with GdnHCl is also manifested in the enormous depression of the transition temperature delta t1/2 (= 18 degrees C) when the GdnHCl concentration is increased from 0 to 1 M. "Regular" proteins experience an average decrease in the transition temperature of 8 +/- 2 degrees C per 1 M change in the concentration of GdnHCl. Study holds ProTherm entries: 8809, 8810, 8811, 8812, 8813, 8814, 8815, 8816 Extra Details: membrane binding protein; thermodynamic equilibrium parameters;,hinge-bending motions; pore formation; heat capacity

Submission Details

ID: NF8wfyk74

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:37 p.m.

Version: 1

Publication Details
Vogl T;Jatzke C;Hinz HJ;Benz J;Huber R,Biochemistry (1997) Thermodynamic stability of annexin V E17G: equilibrium parameters from an irreversible unfolding reaction. PMID:9048549
Additional Information

Study Summary

Number of data points 22
Proteins Annexin A5 ; Annexin A5
Unique complexes 2
Assays/Quantities/Protocols Experimental Assay: dG_H2O ; Experimental Assay: dHcal pH:4.0, ionic:: , buffers:Sodium phosphate: 20 mM ; Experimental Assay: Tm pH:4.0, ionic:: , buffers:Sodium phosphate: 20 mM ; Experimental Assay: dHvH pH:4.0, ionic:: , buffers:Sodium phosphate: 20 mM ; Experimental Assay: dHcal pH:5.0, ionic:: , buffers:Sodium phosphate: 20 mM ; Experimental Assay: Tm pH:5.0, ionic:: , buffers:Sodium phosphate: 20 mM ; Experimental Assay: dHvH pH:5.0, ionic:: , buffers:Sodium phosphate: 20 mM ; Experimental Assay: dHcal pH:6.0, ionic:: , buffers:Sodium phosphate: 20 mM ; Experimental Assay: Tm pH:6.0, ionic:: , buffers:Sodium phosphate: 20 mM ; Experimental Assay: dHvH pH:6.0, ionic:: , buffers:Sodium phosphate: 20 mM ; Experimental Assay: dHcal ionic:: , buffers:Sodium phosphate: 20 mM, pH:7.0 ; Experimental Assay: Tm ionic:: , buffers:Sodium phosphate: 20 mM, pH:7.0 ; Experimental Assay: dHvH ionic:: , buffers:Sodium phosphate: 20 mM, pH:7.0 ; Experimental Assay: dHcal ionic:: , buffers:Sodium phosphate: 20 mM, pH:8.0 ; Experimental Assay: Tm ionic:: , buffers:Sodium phosphate: 20 mM, pH:8.0 ; Experimental Assay: dHvH ionic:: , buffers:Sodium phosphate: 20 mM, pH:8.0 ; Experimental Assay: dHcal buffers:Tris: 50 mM, ionic:KCl: 100 mM, pH:8.0 ; Experimental Assay: Tm buffers:Tris: 50 mM, ionic:KCl: 100 mM, pH:8.0 ; Experimental Assay: dHvH buffers:Tris: 50 mM, ionic:KCl: 100 mM, pH:8.0
Libraries Mutations for sequence MAQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESILTLLTSRSNAQRQEISAAFKTLFGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHALKGAGTNEKVLTEIIASRTPEELRAIKQVYEEEYGSSLEDDVVGDTSGYYQRMLVVLLQANRDPDAGIDEAQVEQDAQALFQAGELKWGTDEEKFITIFGTRSVSHLRKVFDKYMTISGFQIEETIDRETSGNLEQLLLAVVKSIRSIPAYLAETLYYAMKGAGTDDHTLIRVMVSRSEIDLFNIRKEFRKNFATSLYSMIKGDTSGDYKKALLLLCGEDD

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
2IE7 2007-02-06 1.75 Annexin V under 2.0 MPa pressure of nitrous oxide
2IE6 2007-02-06 1.83 Annexin V under 2.0 MPa pressure of xenon
2RAN 1994-11-30 1.89 RAT ANNEXIN V CRYSTAL STRUCTURE: CA2+-INDUCED CONFORMATIONAL CHANGES
1ANX 1994-12-20 1.9 THE CRYSTAL STRUCTURE OF A NEW HIGH-CALCIUM FORM OF ANNEXIN V
1A8B 1998-06-17 1.9 RAT ANNEXIN V COMPLEXED WITH GLYCEROPHOSPHOETHANOLAMINE
1G5N 2001-06-06 1.9 ANNEXIN V COMPLEX WITH HEPARIN OLIGOSACCHARIDES
1A8A 1998-06-17 1.9 RAT ANNEXIN V COMPLEXED WITH GLYCEROPHOSPHOSERINE
1BCY 1998-11-25 1.95 RECOMBINANT RAT ANNEXIN V, T72K MUTANT
1BC3 1998-11-25 1.95 RECOMBINANT RAT ANNEXIN V, TRIPLE MUTANT (T72K, S144K, S228K)
1BC0 1998-11-25 2.0 RECOMBINANT RAT ANNEXIN V, W185A MUTANT
1HVF 1995-03-31 2.0 STRUCTURAL AND ELECTROPHYSIOLOGICAL ANALYSIS OF ANNEXIN V MUTANTS. MUTAGENESIS OF HUMAN ANNEXIN V, AN IN VITRO VOLTAGE-GATED CALCIUM CHANNEL, PROVIDES INFORMATION ABOUT THE STRUCTURAL FEATURES OF THE ION PATHWAY, THE VOLTAGE SENSOR AND THE ION SELECTIVITY FILTER
1HVD 1995-03-31 2.0 STRUCTURAL AND ELECTROPHYSIOLOGICAL ANALYSIS OF ANNEXIN V MUTANTS. MUTAGENESIS OF HUMAN ANNEXIN V, AN IN VITRO VOLTAGE-GATED CALCIUM CHANNEL, PROVIDES INFORMATION ABOUT THE STRUCTURAL FEATURES OF THE ION PATHWAY, THE VOLTAGE SENSOR AND THE ION SELECTIVITY FILTER
1BC1 1998-11-25 2.05 RECOMBINANT RAT ANNEXIN V, QUADRUPLE MUTANT (T72K, S144K, S228K, S303K)
1N41 2003-02-04 2.1 Crystal Structure of Annexin V K27E Mutant
1N42 2003-02-04 2.1 Crystal Structure of Annexin V R149E Mutant
1BCW 1998-11-25 2.1 RECOMBINANT RAT ANNEXIN V, T72A MUTANT
1BCZ 1998-11-25 2.2 RECOMBINANT RAT ANNEXIN V, T72S MUTANT
2H0M 2007-06-19 2.26 Structure of a Mutant of Rat Annexin A5
2XO3 2011-08-03 2.3 Human Annexin V with incorporated Methionine analogue Homopropargylglycine
1HVE 1995-03-31 2.3 STRUCTURAL AND ELECTROPHYSIOLOGICAL ANALYSIS OF ANNEXIN V MUTANTS. MUTAGENESIS OF HUMAN ANNEXIN V, AN IN VITRO VOLTAGE-GATED CALCIUM CHANNEL, PROVIDES INFORMATION ABOUT THE STRUCTURAL FEATURES OF THE ION PATHWAY, THE VOLTAGE SENSOR AND THE ION SELECTIVITY FILTER
1AVR 1994-01-31 2.3 CRYSTAL AND MOLECULAR STRUCTURE OF HUMAN ANNEXIN V AFTER REFINEMENT. IMPLICATIONS FOR STRUCTURE, MEMBRANE BINDING AND ION CHANNEL FORMATION OF THE ANNEXIN FAMILY OF PROTEINS
1AVH 1994-01-31 2.3 CRYSTAL AND MOLECULAR STRUCTURE OF HUMAN ANNEXIN V AFTER REFINEMENT. IMPLICATIONS FOR STRUCTURE, MEMBRANE BINDING AND ION CHANNEL FORMATION OF THE ANNEXIN FAMILY OF PROTEINS
1ANW 1994-12-20 2.4 THE EFFECT OF METAL BINDING ON THE STRUCTURE OF ANNEXIN V AND IMPLICATIONS FOR MEMBRANE BINDING
1SAV 1998-05-27 2.5 HUMAN ANNEXIN V WITH PROLINE SUBSTITUTION BY THIOPROLINE
2H0L 2007-06-19 2.59 Crystal Structure of a Mutant of Rat Annexin A5
2H0K 2007-06-19 2.76 Crystal Structure of a Mutant of Rat Annexin A5
2XO2 2011-08-03 2.8 Human Annexin V with incorporated Methionine analogue Azidohomoalanine
1HAK 1999-02-16 3.0 CRYSTAL STRUCTURE OF RECOMBINANT HUMAN PLACENTAL ANNEXIN V COMPLEXED WITH K-201 AS A CALCIUM CHANNEL ACTIVITY INHIBITOR
1HVG 1995-03-31 3.0 STRUCTURAL AND ELECTROPHYSIOLOGICAL ANALYSIS OF ANNEXIN V MUTANTS. MUTAGENESIS OF HUMAN ANNEXIN V, AN IN VITRO VOLTAGE-GATED CALCIUM CHANNEL, PROVIDES INFORMATION ABOUT THE STRUCTURAL FEATURES OF THE ION PATHWAY, THE VOLTAGE SENSOR AND THE ION SELECTIVITY FILTER
1N44 2003-02-04 3.0 Crystal Structure of Annexin V R23E Mutant

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.8 Annexin A5 P14668 ANXA5_RAT
93.7 Annexin A5 P48036 ANXA5_MOUSE
95.9 Annexin A5 P81287 ANXA5_BOVIN
98.4 Annexin A5 Q4R4H7 ANXA5_MACFA
100.0 Annexin A5 Q5R1W0 ANXA5_PANTR
100.0 Annexin A5 P08758 ANXA5_HUMAN