pH-induced changes of the structure of small heat shock proteins with molecular mass 24/27 kDa (HspB1) q Ivan S. Chernik a , Olesya O. Panasenko a , Yi Li a , Steven B. Marston b , Nikolai B. Gusev a, * a Department of Biochemistry, School of Biology, Moscow State University, Moscow 119992, Russia b Imperial College, School of Medicine at National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK Received 20 September 2004 Available online 12 October 2004 Abstract The effect of pH on the structure of recombinant chicken Hsp24, human Hsp27 and their 3D mutants mimicking phosphoryla- tion at Ser15, Ser77/78, and Ser81/82 was analyzed. Circular dichroism and fluorescent spectroscopy indicate that changes of pH in the range 6.0–7.5 weakly affected the secondary and tertiary structure of the wild type proteins, but induced noticeable changes in the structure of their 3D mutants. According to size-exclusion chromatography and analytical ultracentrifugation variation of pH- induced pronounced changes in the quaternary structure of small heat shock proteins and acidification resulted in accumulation of large oligomers of Hsp24/27. It is concluded that small changes of pH strongly affect the quaternary structure of small heat shock proteins and by this means can influence their functioning in the cell. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Small heat shock proteins; Structure; Phosphorylation; Acidosis Small heat shock proteins (sHsp), having low molec- ular mass (15–40 kDa) and containing so-called a-crys- tallin domain, form a widespread group of stress proteins [1–3]. b-Sheets of the a-crystallin domain lo- cated in the C-terminal part of sHsp are involved in for- mation of stable dimers [4,5]. Variable N-terminal domains provide for formation of high molecular mass oligomers consisting of 4–32 monomers [1,4–6]. These high molecular mass oligomers are in concentration dependent rapid equilibrium with dimers or low molec- ular mass oligomers of small heat shock proteins [6–8]. Different protein kinases phosphorylate small heat shock proteins and by this means affect their quaternary structure and chaperone activity [1,2,8]. Expression of sHsp is increased in response to differ- ent kinds of injury and their content is especially high in heart, striated, and smooth muscle [1,9]. Overexpression of small heat shock proteins protects the cell from heat shock [6], oxidative stress [8,10], energy depletion, and other unfavorable conditions [1]. Recently, published data indicate that small heat shock proteins participate in protection of the cells against ischemia [11–13]. Ische- mia followed by reperfusion is accompanied by oxida- tive stress and acidosis when intracellular pH drops by 1 or even 1.5 U of pH [14,15]. The protective effect of sHsp during oxidative stress was analyzed in several publications [3,8,10], however, to our knowledge except of one brief report [15], the behavior of sHsp during aci- dosis remains practically uncharacterized. Therefore, this paper deals with analysis of pH-induced changes 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.09.176 q Abbreviations: bis-ANS, 4,4 0 -bis(1-anilinonaphthalene-8-sulfo- nate); CD, circular dichroism; FRET, fluorescence resonance energy transfer; Hsp24, recombinant chicken heat shock protein with appar- ent molecular mass 24 kDa; Hsp27, recombinant human heat shock protein with apparent molecular mass 27 kDa; 3D mutants of Hsp24 or Hsp27, mutants with replacing S15, S77, S81 (Hsp24) or S15, S78, S82 (Hsp27) with aspartic acid; PMSF, phenylmethanesulfonyl fluo- ride; sHsp, small heat shock protein(s); WT, wild type. * Corresponding author. Fax: +7 095 939 2747. E-mail address: NBGusev@mail.ru (N.B. Gusev). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 324 (2004) 1199–1203 BBRC