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
1ANW 1993-10-26T00:00:00+0000 2.4 THE EFFECT OF METAL BINDING ON THE STRUCTURE OF ANNEXIN V AND IMPLICATIONS FOR MEMBRANE BINDING
1ANX 1993-10-26T00:00:00+0000 1.9 THE CRYSTAL STRUCTURE OF A NEW HIGH-CALCIUM FORM OF ANNEXIN V
1AVH 1991-10-17T00:00:00+0000 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
1AVR 1991-10-17T00:00:00+0000 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
1HAK 1997-12-10T00:00:00+0000 3.0 CRYSTAL STRUCTURE OF RECOMBINANT HUMAN PLACENTAL ANNEXIN V COMPLEXED WITH K-201 AS A CALCIUM CHANNEL ACTIVITY INHIBITOR
1HVD 1994-06-29T00:00:00+0000 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
1HVE 1994-06-29T00:00:00+0000 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
1HVF 1994-06-29T00:00:00+0000 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
1HVG 1994-06-29T00:00:00+0000 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
1SAV 1997-11-24T00:00:00+0000 2.5 HUMAN ANNEXIN V WITH PROLINE SUBSTITUTION BY THIOPROLINE

Relevant UniProtKB Entries

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