Tài liệu Báo cáo khoa học: Local stability identification and the role of a key aromatic amino acid

Local stability identification and the role of a key aromatic

amino acid residue in staphylococcal nuclease refolding

Zhengding Su3

, Jiun-Ming Wu1

, Huey-Jen Fang1

, Tian-Yow Tsong2,3 and Hueih-Min Chen1

1 Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan, ROC

2 Institute of Physics, Academia Sinica, Taipei, Taiwan, ROC

3 Department of Biochemistry, University of Minnesota College of Biological Sciences, St Paul, MN, USA

Staphylococcal nuclease (SNase) is a single-domain

protein of 149 amino acids. Its 3D structure has been

examined by NMR [1–3] and X-ray crystallography

[4,5]. However, only part of the structure (positions 1–

141) can be confirmed. The segment 142–149 has not

been defined with certainty because of its apparent

flexibility. Structural observations made with NMR or

X-ray have led to the prediction that certain amino

acid(s) in the flexible segment stabilize the rest of the

structure, in particular the key amino acid(s) located

close to this flexible segment. The tryptophan at posi￾tion 140, for example, may play an important role in

maintaining the protein structure formed by amino

acids 1–141. In this study, we used site-directed muta￾genesis to generate point mutations and truncations

around this position to explore the above prediction.

As shown by Chen and colleagues [6], SNase protein

can be unfolded by lowering its pH (for example, from

pH 7 to pH 2). About 2.5 protons are associated with

the key glutamic amino acid residues at positions 75 and

129. This association between protons and key amino

acids leads the protein to unfold. However, the refolding

process may be different and more complex because the

transition of refolding is from many unfolded states to

a single native state. Based on previous kinetic experi￾ments using single-jump and double-jump stopped-flow

for refolding [7,8], SNase protein can be refolded in vitro

to its active 3D conformation in milliseconds. The

sequence of equilibrium reactions between the three

denatured states and one native state can be shown as

[9] N « D1 « D2 « D3, where N is the protein in its

native state and Di (i ¼ 1–3) indicates the protein in its

unfolded state. This scheme has been used to solve puz￾zles such as accumulated intermediates and to conduct

random searches among ‘microscopic states’ [8]. The

early stages of refolding (Di fi N) occur via key amino

acid(s) which act as nucleation centres before proton

dissociation. Subsequently, these centres trigger the

condensation of random polypeptide chains into the

compact form of the native state.

In this study, the effects of mutating W140 [10]

on SNase protein conformation and stability were

Keywords

aromatic amino acid; refolding; stability;

staphylococcal nuclease

Correspondence

H-M. Chen, Institute of BioAgricultural

Sciences, Academia Sinica, Taipei,

Taiwan 115, R.O.C.

Fax: +886 2 2788 8401

Tel: +886 2 2785 5696 ext. 8030

E-mail: [email protected]

(Received 3 May 2005, revised 3 June

2005, accepted 13 June 2005)

doi:10.1111/j.1742-4658.2005.04814.x

Staphylococcal nuclease (SNase) is a model protein that contains one

domain and no disulfide bonds. Its stability in the native state may be

maintained mainly by key amino acids. In this study, two point-mutated

proteins each with a single base substitution [alanine for tryptophan

(W140A) and alanine for lysine (K133A)] and two truncated fragment

proteins {positions 1–139 [SNase(1–139) or W140O] and positions 1–141

[SNase(1–141) or E142O]} were generated. Differential scanning micro￾calorimetry in thermal denaturation experiments showed that K133A and

E142O have nearly unchanged DHcal relative to the wild-type, whereas

W140A and W140O display zero enthalpy change (DHcal
0). Far-UV CD

measurements indicate secondary structure in W140A but not W140O, and

near-UV CD measurements indicate no tertiary structure in either W140

mutant. These observations indicate an unusually large contribution of

W140 to the stability and structural integrity of SNase.

Abbreviations

DSC, differential scanning calorimetry; SNase, staphylococcal nuclease.

3960 FEBS Journal 272 (2005) 3960–3966 ª 2005 FEBS