Tài liệu Báo cáo Y học: Biphasic reductive unfolding of ribonuclease A is temperature dependent pdf

Biphasic reductive unfolding of ribonuclease A is temperature

dependent

Yong-Bin Yan1,2, Ri-Qing Zhang1,2 and Hai-Meng Zhou3

1

NMR Laboratory, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China; 2

State Key

Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua

University, Beijing, China; 3

Protein Science Laboratory of the Ministry of Education, Department of Biological Sciences and

Biotechnology, Tsinghua University, Beijing, China

The kinetics of the reversible thermal unfolding, irreversible

thermal unfolding, and reductive unfolding processes of

bovine pancreatic ribonuclease A (RNase A) were investi￾gated in NaCl/Pi solutions. Image parameters including

Shannon entropy, Hamming distance, mutual information

and correlation coefficient were used in the analysis of the

CD and 1D NMR spectra. The irreversible thermal

unfolding transition of RNase A was not a cooperative

process, pretransitional structure changes occur before the

main thermal denaturation. Different dithiothreitol (dithio￾threitolred) concentration dependencies were observed

between 303 and 313 K during denaturation induced by a

small amount of reductive reagent. The protein selectively

follows a major unfolding kinetics pathway with the selec￾tivity can be altered by temperature and reductive reagent

concentration. Two possible explanations of the selectivity

mechanism were discussed.

Keywords: image analysis; proton nuclear magnetic reson￾ance; reductive unfolding; thermal unfolding; unfolding

kinetics.

Dynamic analysis of the unfolding and refolding pathways

and identification of the specific conformational changes

which form the individual intermediates involved in the

rate-limited pathway(s) can distinguish one pathway from

another and play fundamental importance for protein

folding [1,2]. It is usually of considerable interest to estimate

the conformational changes of both the whole protein

tertiary structure and of specific sites observed by spectro￾scopic techniques in different redox systems and solvent

conditions. Protein unfolding is highly pertinent to protein

folding [3] and is more controllable for more comprehensive

study by slowing the unfolding process carried out at

physiological pH and temperature [4]. Such studies in turn

provide new insights into the functional properties and

mechanisms of proteins, which will lead to a more detailed

and more complete description of biological functions [5].

Bovine pancreatic ribonuclease A (RNase A; EC

3.1.27.5) contains 124 residues with four native disulfide

bonds (Cys26–Cys84, Cys40–Cys95, Cys58–Cys110, and

Cys65–Cys72). RNase A has played a crucial role as a

model system in studies of protein structure, folding,

stability, and chemistry [6]. It folds and unfolds through

multiple pathways, with the rate-limiting steps in the well￾populated pathways involving the formation of distinct

transition intermediates [1–3,7,8]. The complexity of the

multiple pathways means that different mechanisms may

occur with different types of redox systems and different

solvent conditions [9,10]. Thus a comprehensive investiga￾tion under different conditions using different methods is

essential to elucidate the protein folding and unfolding

processes.

Much effort has been devoted in recent years to

understanding the mechanism and the main factors that

control protein folding, and to developing approaches that

allow researchers to investigate the multifarious aspects of

protein folding and unfolding. While the determination of

the protein structural transitions that occur in the pathways

is at the heart of studies on unfolding and refolding

processes [10], dynamic analysis of the different processes is

necessary to evaluate the different pathways. These studies

will also clarify the key factor(s) controlling the processes

and clarify the unfolding and refolding mechanism associ￾ated with the redox properties and solvent conditions.

Therefore, we have used 1

H NMR spectra to investigate the

unfolding dynamics of RNase A during denaturation by

different concentrations of reductive reagent dithiothreitol

(dithiothreitolred) at different temperatures. Similar studies

were carried out by Rothwarf and Scheraga [10] in which

the temperature dependence of RNase A regeneration was

studied with dithiothreitolox/dithiothreitolred and GSSG/

GSH systems. Their results suggested that the regeneration

process with the two types of redox reagents proceeded

through different pathways with significantly different

temperature dependencies. Here we present a temperature

Correspondence to Y.-B. Yan, Department of Biological Sciences

and Biotechnology, Tsinghua University, Beijing 100084, China.

E-mail: [email protected] or H.-M. Zhou, Department of

Biological Sciences and Biotechnology, Tsinghua University,

Beijing 100084, China. E-mail: [email protected]

Abbreviations: C, Correlation coefficient; des-[40–95], RNase A

lacking the 40–95 disulfide bond; des-[65–72], RNase A lacking the

65–72 disulfide bond; DSS, 2,2-dimethyl-2-sila-pentanesulfonate;

FID, free induction decay; GSH, reduced glutathione; GSSG,

oxidized glutathione; H, Shannon entropy; HD, Hamming distance;

MI, Shannon mutual information; RNase A, Bovine pancreatic

ribonuclease A.

(Received 23 June 2002, revised 3 September 2002,

accepted 11 September 2002)

Eur. J. Biochem. 269, 5314–5322 (2002)
FEBS 2002 doi:10.1046/j.1432-1033.2002.03251.x