Site-Directed Mutagenesis of Myoglobin for Studies of Their Interaction with Iron(III) by Multi-Spectroscopic Techniques

Qian Tang,Jing Ma,Xue-Fang Zheng,Xiao-Jun Peng,Yan-Jie Yang,Hong-Yu Cao,Jing-Yun Wang

doi:10.1155/2013/314345

Qian Tang, Jing Ma + Show 5 more

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https://doi.org/10.1155/2013/314345

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Abstract

In order to investigate how the amino acids on the surface of myoglobin molecule influence myoglobin's structure and function, a variety of spectroscopy techniques were applied in the study of the interaction between Fe(III) and myoglobin (wild type and its mutants, D44K, D60K, and K56D). The results demonstrate that Fe(III) can quench the fluorescence of wild type and mutants of myoglobin, and the quenching mechanisms are static quenching. It is found that the binding distance between Fe(III) and myoglobin mutants gets smaller, the binding capacity increases by the values of binding constant and the bimolecular quenching constant as well as the binding distance. Those data also indicate that the metal ion Fe(III) can interact strongly with myoglobin mutants. The three-dimensional conformation change after surface amino acids are replaced is detected by the UV absorption spectroscopy and fluorescence spectroscopy, which make mutants become more dynamic and change its function and interaction with Fe(III) strongly.

Highlights

Proteins have a variety of important physiological functions. ey participate in every reaction of the living body and are essential for the translation of the genetic code of life, transcription of the information, and replication of DNA
In order to investigate how the amino acids on the surface of myoglobin molecule in uence myoglobin’s structure and function, a variety of spectroscopy techniques were applied in the study of the interaction between Fe(III) and myoglobin. e results demonstrate that Fe(III) can quench the uorescence of wild type and mutants of myoglobin, and the quenching mechanisms are static quenching
It is found that the binding distance between Fe(III) and myoglobin mutants gets smaller, the binding capacity increases by the values of binding constant and the bimolecular quenching constant as well as the binding distance. ose data indicate that the metal ion Fe(III) can interact strongly with myoglobin mutants. e three-dimensional conformation change a er surface amino acids are replaced is detected by the UV absorption spectroscopy and uorescence spectroscopy, which make mutants become more dynamic and change its function and interaction with Fe(III) strongly

Summary

Introduction

Proteins have a variety of important physiological functions. ey participate in every reaction of the living body and are essential for the translation of the genetic code of life, transcription of the information, and replication of DNA. Metal ions exist in biological uids in various forms and interact with biomolecules in many ways, which perform kinds of functions in organism, such as electron transfer, oxygen carrier, and enzyme active center [1]. Due to its simple structure and important biological activity, it has been the model protein for the study of proteins’ interaction with their ligands. In order to clarify the impact of speci c amino acids residues on the surface of Mb on the structure and function of myoglobin, in the present study, PCR site-directed mutagenesis technique was used to obtain myoglobin mutants D44K, D60K, and K56D and a variety of spectrographic methods were adopted to study the interactions between metal ion Fe(III) and myoglobins (wild type and mutants). T 1: e bimolecular quenching constant (KKqq), the dynamic quenching constants (KKssss), the correlation coefficients (RR) in SternVolmer curve

Experimental

Experimental Methods

Result and Discussion