Protein Science zyxwvutsrqponm (1997), 6:2385-2396. Cambridge University Press. Printed in the USA Copyright zyxwvutsrqp 0 1997 The Protein Society Domain organization of calbindin D28k as determined from the association of six synthetic EF-hand fragments zyxw SARA LINSE,' EVA THULIN,' LIDA K. GIFFORD? DINA RADZEWSKY,* JAMES HAG AN,^ ROSEMARIE R. WILK,~ AND KARIN s. AKERFELDT~ 'Physical Chemistry 2, Chemical Centre, University of Lund, S-221 00 Lund, Sweden 'Department of Chemistry, Rutgers University, Camden, New Jersey 08102 'DuPont Merck Pharmaceutical Co., Wilmington, Delaware 19880-0500 (RECEIVED February 24, 1997; ACCEPTED July 7, 1997) Abstract Calbindin DZsk is an intracellular Ca*+-binding protein containing six subdomains of EF-hand type. The number and identity of the globular domains within this protein have been elucidated using six synthetic peptide fragments, each corresponding to one EF-hand subdomain. All six peptides were mixed in equimolar amounts in the presence of 10 mM Ca2+ to allow for the reconstitution of domains. The mixture was compared to native calbindin D28k and to the sum of the properties of the individual peptides using circular dichroism (CD), fluorescence, and 'H NMR spectroscopy, as well as gel filtration and ion-exchange chromatography. It was anticipated that if the peptides associate to form native-like domains, the properties would zyxwvutsrq be similar to those of the intact protein, whereas if they did not interact, they would be the same as the properties of the isolated peptides. The results show that the peptides in the mixture interact with one another. For example, the CD and fluorescence spectra for the mixture are very similar to those of the intact calbindin D2skrsuggesting that the mixed EF-hand fragments associate to form a native-like structure. To determine the number of domains and the subdomain composition of each domain in calbindin D28k, a variety of peptide combinations containing two to five EF-hand fragments were studied. The spectral and chromatographic properties of all the mixtures containing less than six peptides were closer to the sum of the properties of the relevant individual peptides than to the mixture of the six peptides. The results strongly suggest that all six EF-hands are packed into one globular domain. The association of the peptide fragments is observed to drive the folding of the individual subdomains. For example, one of the fragments, EF2, which is largely unstructured in isolation even in the presence of high concentrations of Ca2+,is considerably more structured in the presence of the other peptides, as judged by CD difference spectroscopy. The CD data also suggest that the packing between the individual subdomains is specific. Keywords: calbindin D28k; Ca2+-binding protein; domain organization; EF-hand; protein folding; spectroscopy; subdomain association; synthetic peptides. The EF-hand (Kretsinger & Nockolds, 1973) is a highly conserved helix-loop-helix structural motif present in a wide variety of calcium- binding proteins whose functions are to act as calcium buffers or to regulate specific target proteins in a calcium-dependent manner. To date, hundreds of EF-hands have been identified through se- quence homology (Kretsinger 1987; Nakayama et al., 1992). One EF-hand is referred to as a subdomain. It is the packing arrange- ment of two or more EF-hand subdomains into a functional do- main that confers the specific biological functions to the individual protein. Many residues in the helix-loop-helix motif are highly con- served, particularly in the calcium-binding loop (Fig. 1). Although there exists a high degree of sequence homology among the EF- hands, they have been found to associate into different types of domain structures, typically containing between two and eight EF- hand subdomains associated into one or more globular domains. Due to interactions between the subdomains within a domain. the binding of Ca2+ typically occurs with positive cooperativity. The Reprint requests to: Sara Linse; Physical Chemistry 2, Lund University, smallest type of domain consists of two EF-hands packed into a four-helix bundle. The Ca*+-binding loops, which are positioned Abbreviations: NMR, nuclear magnetic resonance: CD. circular dichro- at the Same end of the bundle, form a short 8-sheet. One domain Chemical Centre, P.O. Box 124, S-221 00 Lund, Sweden; e-mail: Sara@ bor.fkem2.lth.se or to Karin S. Akerfeldt; akerfeld@crab.rutgers.edu. zyxwvuts I ism; FPLC, fast protein liquid chromatography , , of this type is present in calbindin Dgk(Szebenyi & Moffat, 1986; z 2385