Modified spectrophotometer for multi-dimensional circular dichroism/fluorescence data acquisition in titration experiments: application to the pH and guanidine-HCI induced unfolding of apomyoglobin.


Abstract

In a previous paper (Ramsay and Eftink, Biophys. J. 66:516-523) we reported the development of a modified spectrophotometer that can make nearly simultaneous circular dichroism (CD) and fluorescence measurements. This arrangement allows multiple data sets to be collected during a single experiment, resulting in a saving of time and material, and improved correlation between the different types of measurements. The usefulness of the instrument was shown by thermal melting experiments on several different protein systems. This CD/fluorometer spectrophotometer has been further modified by interfacing with a syringe pump and a pH meter. This arrangement allows ligand, pH, and chemical denaturation titration experiments to be performed while monitoring changes in the sample's CD, absorbance, fluorescence, and light scattering properties. Our data acquisition program also has an ability to check whether the signals have approached equilibrium before the data is recorded. For performing pH titrations we have developed a procedure which uses the signal from a pH meter in a feedback circuit in order to collect data at evenly spaced pH intervals. We demonstrate the use of this instrument with studies of the unfolding of sperm whale apomyoglobin, as induced by acid pH and by the addition of guanidine-HCI. Study holds ProTherm entries: 7408, 7409, 7410, 7411 Extra Details: the transition is from native to intermediate thermal melting experiment; ligand; chemical denaturation;,data acquisition program

Submission Details

ID: ariYMUGp

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:34 p.m.

Version: 1

Publication Details
Ramsay G;Ionescu R;Eftink MR,Biophys. J. (1995) Modified spectrophotometer for multi-dimensional circular dichroism/fluorescence data acquisition in titration experiments: application to the pH and guanidine-HCI induced unfolding of apomyoglobin. PMID:8527683
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
6BMG 2017-11-14T00:00:00+0000 1.88 Structure of Recombinant Dwarf Sperm Whale Myoglobin (Oxy)
5YCI 2017-09-07T00:00:00+0000 1.97 Ancestral myoglobin aMbWb' of Basilosaurus relative (polyphyly)
5YCJ 2017-09-07T00:00:00+0000 1.58 Ancestral myoglobin aMbWb' of Basilosaurus relative (polyphyly) imidazole-ligand
101M 1997-12-13T00:00:00+0000 2.07 SPERM WHALE MYOGLOBIN F46V N-BUTYL ISOCYANIDE AT PH 9.0
102M 1997-12-15T00:00:00+0000 1.84 SPERM WHALE MYOGLOBIN H64A AQUOMET AT PH 9.0
103M 1997-12-16T00:00:00+0000 2.07 SPERM WHALE MYOGLOBIN H64A N-BUTYL ISOCYANIDE AT PH 9.0
104M 1997-12-18T00:00:00+0000 1.71 SPERM WHALE MYOGLOBIN N-BUTYL ISOCYANIDE AT PH 7.0
105M 1997-12-18T00:00:00+0000 2.02 SPERM WHALE MYOGLOBIN N-BUTYL ISOCYANIDE AT PH 9.0
106M 1997-12-21T00:00:00+0000 1.99 SPERM WHALE MYOGLOBIN V68F ETHYL ISOCYANIDE AT PH 9.0
107M 1997-12-22T00:00:00+0000 2.09 SPERM WHALE MYOGLOBIN V68F N-BUTYL ISOCYANIDE AT PH 9.0

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
90.3 Myoglobin P02180 MYG_BALPH
90.8 Myoglobin P68278 MYG_PHOPH
90.8 Myoglobin P68277 MYG_PHODA
90.8 Myoglobin P68279 MYG_TURTR
90.8 Myoglobin P68276 MYG_DELDE
90.8 Myoglobin Q0KIY7 MYG1_STEAT
91.4 Myoglobin P02173 MYG_ORCOR
90.9 Myoglobin P02179 MYG_BALAC
92.1 Myoglobin P02174 MYG_GLOME
91.4 Myoglobin P02181 MYG_INIGE
91.4 Myoglobin Q0KIY3 MYG_PENEL
92.2 Myoglobin P02178 MYG_MEGNO
92.9 Myoglobin P02177 MYG_ESCRO
92.9 Myoglobin Q0KIY2 MYG_BALED
92.9 Myoglobin Q0KIY1 MYG_BALBO
96.8 Myoglobin P02184 MYG_KOGSI
96.8 Myoglobin Q0KIY5 MYG_KOGBR
100.0 Myoglobin P02185 MYG_PHYMC
90.1 Myoglobin P02182 MYG_ZIPCA
90.1 Myoglobin Q0KIY0 MYG_MESST
90.1 Myoglobin P02183 MYG_MESCA