Study of strong to ultratight protein interactions using differential scanning calorimetry.


Abstract

Data from differential scanning calorimetry (DSC) may be used to estimate very large binding constants that cannot be conveniently measured by more conventional equilibrium techniques. Thermodynamic models have been formulated to describe interacting systems that involve either one thermal transition (protein-ligand) or two thermal transitions (protein-protein) and either 1:1 or higher binding stoichiometry. Methods are described for obtaining binding constants and heats of binding by two different methods: calculation or simulation fitting of data. Extensive DSC data on 2'CMP binding to RNase are presented and analyzed by the two methods. It is found that the methods agree when binding sites are completely saturated, but substantial errors arise in the calculation method when site saturation is incomplete and the transition of liganded molecules overlaps that of unliganded molecules. This arises primarily from an inability to determine TM (i.e., the temperature where concentrations of folded and unfolded protein are equal) under weak-binding conditions. Results from simulation show that the binding constants and heats of binding from the DSC method agree quantitatively with corresponding estimates obtained from equilibrium methods when extrapolated to the same temperature. It was also found from the DSC data that the binding constant decreases with increasing concentration of ligand, which might arise from nonideality effects associated with dimerization of 2'CMP. Simulations show that the DSC method is capable of estimating binding constants for ultratight interactions up to perhaps 10(40) M-1 or higher, while most equilibrium methods fail well below 10(10) M-1. DSC data from the literature on a number of interacting systems (trypsin-soybean trypsin inhibitor, trypsin-ovomucoid, trypsin-pancreatic trypsin inhibitor, chymotrypsin-subtilisin inhibitor, subtilisin BPN-subtilisin inhibitor, RNase S protein-RNase S peptide, avidin-biotin, ovotransferrin-Fe3+, superoxide dismutase-Zn2+, alkaline phosphatase-Zn2+, and assembly of regulatory and catalytic subunits of aspartate transcarbamoylase) were analyzed by simulation fitting or by calculation. Apparent single-site binding constants ranged from ca. 10(5) to 10(20) M-1, while the interaction constant for assembly of aspartate transcarbamoylase was estimated as 10(37) in molarity units. For most of these systems, the DSC interaction constants compared favorably with other literature estimates, for some it did not for reasons unknown, while for still others this represented the first estimate. Simulations show that for proteins having two binding sites for the same ligand within a single cooperative unit, ligand rearrangement will occur spontaneously during a DSC scan as the transition temperature of the unliganded protein is approached.(ABSTRACT TRUNCATED AT 400 WORDS) Study holds ProTherm entries: 4014 Extra Details: binding constants; thermodynamic models; thermal transition;,ligand rearrangement; low-temperature configuration

Submission Details

ID: cxgEuQWg

Submitter: Connie Wang

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

Version: 1

Publication Details
Brandts JF;Lin LN,Biochemistry (1990) Study of strong to ultratight protein interactions using differential scanning calorimetry. PMID:2204424
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
6QMN 2019-02-07T00:00:00+0000 2.31 Crystal structure of a Ribonuclease A-Onconase chimera
1A2W 1998-01-12T00:00:00+0000 2.1 CRYSTAL STRUCTURE OF A 3D DOMAIN-SWAPPED DIMER OF BOVINE PANCREATIC RIBONUCLEASE A
1A5P 1998-02-17T00:00:00+0000 1.6 C[40,95]A VARIANT OF BOVINE PANCREATIC RIBONUCLEASE A
1A5Q 1998-02-17T00:00:00+0000 2.3 P93A VARIANT OF BOVINE PANCREATIC RIBONUCLEASE A
1AFK 1997-03-08T00:00:00+0000 1.7 CRYSTAL STRUCTURE OF RIBONUCLEASE A IN COMPLEX WITH 5'-DIPHOSPHOADENOSINE-3'-PHOSPHATE
1AFL 1997-03-08T00:00:00+0000 1.7 RIBONUCLEASE A IN COMPLEX WITH 5'-DIPHOSPHOADENOSINE 2'-PHOSPHATE AT 1.7 ANGSTROM RESOLUTION
1AFU 1997-03-14T00:00:00+0000 2.0 STRUCTURE OF RIBONUCLEASE A AT 2.0 ANGSTROMS FROM MONOCLINIC CRYSTALS
1AQP 1997-07-31T00:00:00+0000 2.0 RIBONUCLEASE A COPPER COMPLEX
1B6V 1999-01-18T00:00:00+0000 2.0 CRYSTAL STRUCTURE OF A HYBRID BETWEEN RIBONUCLEASE A AND BOVINE SEMINAL RIBONUCLEASE
1BEL 1995-12-21T00:00:00+0000 1.6 HYDROLASE PHOSPHORIC DIESTER, RNA

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
96.0 Ribonuclease pancreatic Q29606 RNAS1_ORYLE
90.3 Ribonuclease pancreatic P00662 RNAS1_GIRCA
92.7 Ribonuclease pancreatic P00668 RNAS1_ANTAM
95.2 Ribonuclease pancreatic P00660 RNAS1_CONTA
93.5 Ribonuclease pancreatic P07848 RNAS1_EUDTH
96.0 Ribonuclease pancreatic P07847 RNAS1_AEPME
95.2 Ribonuclease pancreatic P00657 RNAS1_BUBBU
96.8 Ribonuclease pancreatic P67927 RNAS1_SHEEP
96.8 Ribonuclease pancreatic P67926 RNAS1_CAPHI
100.0 Ribonuclease pancreatic P61823 RNAS1_BOVIN
100.0 Ribonuclease pancreatic P61824 RNAS1_BISBI