Andrzej Bierzyński

Affinity of S100A1 protein for calcium increases dramatically upon glutathionylation

S100A1 is a typical representative of a group of EF‐hand calcium‐binding proteins known as the S100 family. The protein is composed of two α subunits, each containing two calcium‐binding loops (N and C). At physiological pH (7.2) and NaCl concentration (100 mm), we determined the microscopic binding constants of calcium to S100A1 by analysing the Ca2+‐titration curves of Trp90 fluorescence for both the native protein and its Glu32 → Gln mutant with an inactive N‐loop. Using a chelator method, we also determined the calcium‐binding constant for the S100A1 Glu73 → Gln mutant with an inactive C‐loop. The protein binds four calcium ions in a noncooperative way with binding constants of K1 =4 ± 2 × 103 m−1 (C‐loops) and K2≈ 102 m−1 (N‐loops). Only when both loops are saturated with calcium does the protein change its global conformation, exposing to the solvent hydrophobic patches, which can be detected by 2‐p‐toluidinylnaphthalene‐6‐sulfonic acid – a fluorescent probe of protein‐surface hydrophobicity. S‐Glutathionylation of the single cysteine residue (85) of the α subunits leads to a 10‐fold increase in the affinity of the protein C‐loops for calcium and an enormous – four orders of magnitude – increase in the calcium‐binding constants of its N‐loops, owing to a cooperativity effect corresponding to ΔΔG = −6 ± 1 kcal·mol−1. A similar effect is observed upon formation of the mixed disulfide with cysteine and 2‐mercaptoethanol. The glutathionylated protein binds TRTK‐12 peptide in a calcium‐dependent manner. S100A1 protein can act, therefore, as a linker between the calcium and redox signalling pathways.

Quantitative fluorescenct analysis of different conformational forms of DNA bound to the dye, 4′,6-diamidine-2-phenylindole, and sepasated by gel electrophoresis

Abstract A quantitative fluorescent method for estimating the amounts of different conformational forms of the same DNA on agarose gels is described. Supercoiled, open circular, and linear forms of PM2 DNA and fluorescent dye (4′,6-diamidine-2-phenylindole) were used. The results are compared with respective radiometric estimations and are shown to be highly reproducible.

Atomic resolution structure of squash trypsin inhibitor: unexpected metal coordination

CMTI-I, a small-protein trypsin inhibitor, has been crystallized as a 4:1 protein-zinc complex. The metal is coordinated in a symmetric tetrahedral fashion by glutamate/glutamic acid side chains. The structure was solved by direct methods in the absence of prior knowledge of the special position metal centre and refined with anisotropic displacement parameters using diffraction data extending to 1.03 A. In the final calculations, the main-chain atoms of low B(eq) values were refined without restraint control. The two molecules in the asymmetric unit have a conformation that is very similar to that reported earlier for CMTI-I in complex with trypsin, despite the Met8Leu mutation of the present variant. The only significant differences are in the enzyme-binding epitope (including the Arg5 residue) and in a higher mobility loop around Glu24. The present crystal structure contains organic solvent molecules (glycerol, MPD) that interact with the inhibitor molecules in an area that is at the enzyme-inhibitor interface in the CMTI-I-trypsin complex. A perfectly ordered residue (Ala18) has an unusual Ramachandran conformation as a result of geometrical strain introduced by the three disulfide bridges that clamp the protein fold. The results confirm deficiencies of some stereochemical restraints, such as peptide planarity or the N-C(alpha)-C angle, and suggest a link between their violations and hydrogen bonding.

Synthesis, cloning and expression in Escherichia coli of a gene coding for the Met8 → Leu CMTI I — a representative of the squash inhibitors of serine proteinases

A chemically synthesized gene coding for a Cucurbita maxima trypsin inhibitor modified at position P′3 (Met8→ Leu CMTI I), i.e. at the third position downstream of the reactive site bond (Arg5‐Ile), was cloned into a derivative of the plasmid pAED4 that utilizes a T7 expression system. The gene was expressed in Escherichia coli as a fusion protein that accumulates in inclusion bodies. After reduction and CNBr cleavage of the fusion protein followed by oxidative refolding and reverse‐phase HPLC, about 5 mg of pure protein was obtained per 1 of cell culture. Association constants of recombinant Leu‐8‐CMTI I with bovine β‐trypsin and human cathepsin G are the same, within experimental error, as for CMTI I isolated from a natural source.

Conformational role of His-12 in C-peptide of ribonuclease A.

Possible interactions of the His-12 ring with other side chain and backbone groups of C-peptide lactone (CPL) are discussed. The works published so far are critically reviewed and compared with the latest results obtained by the authors. The main new conclusion is that in the helical conformation of CPL, the Phe-8 and His-12 rings are clustered together. Studies of Phe-8----Ala analogs of CPL and calculations of ring current effects satisfactorily explain the observed environmental shifts of Phe-8 and His-12 protons in NMR spectra of CPL. Interaction between both rings is favorable for alpha-helix formation, but cannot explain an increase in helix stability related with protonation of His-12. This effect arises from favorable interactions of the charged His+-12 ring with the helix backbone.

Impact of Calcium Binding and Thionylation of S100A1 Protein on Its Nuclear Magnetic Resonance-Derived Structure and Backbone Dynamics

S100 proteins play a crucial role in multiple important biological processes in vertebrate organisms acting predominantly as calcium signal transmitters. S100A1 is a typical representative of this family of proteins. After four Ca(2+) ions bind, it undergoes a dramatic conformational change, resulting in exposure, in each of its two identical subunits, a large hydrophobic cleft that binds to target proteins. It has been shown that abnormal expression of S100A1 is strongly correlated with a number of severe human diseases: cardiomyopathy and neurodegenerative disorders. A few years ago, we found that thionylation of Cys 85, the unique cysteine in two identical S100A1 subunits, leads to a drastic increase of the affinity of the protein for calcium. We postulated that the protein activated by thionylation becomes a more efficient calcium signal transmitter. Therefore, we decided to undertake, using nuclear magnetic resonance methods, a comparative study of the structure and dynamics of native and thionylated human S100A1 in its apo and holo states. In this paper, we present the results obtained for both forms of this protein in its holo state and compare them with the previously published structure of native apo-S100. The main conclusion that we draw from these results is that the increased calcium binding affinity of S100A1 upon thionylation arises, most probably, from rearrangement of the hydrophobic core in its apo form.

LUMINESCENCE OF PEPTIDE-BOUND TERBIUM IONS : DETERMINATION OF BINDING CONSTANTS

Luminescence of Tb3+ ions bound to a calmodulin fragments has been studied. It is shown that during their lifetime excited ions dissociate from the peptide. If concentration of free peptide is high enough they can be coordinated again. As a consequence, observed terbium luminescence lifetime and intensity depends not only on binding equilibrium, but also on concentration or free peptide molecules. In such a system terbium binding constant cannot be correctly determined by simple steady‐state measurements of luminescence intensities. Instead, terbium luminescence decay curves measured at various peptide concentrations must be analysed. Such an analysis has been made for a fragment of the IIIrd calcium binding domain of rat testis calmodulin. Rate constant or terbium association and the equilibrium binding constant corresponding to the best fit of theoretical functions to experimental points have been determined.

Theoretical estimation of the calcium-binding constants for proteins from the troponin C superfamily based on a secondary structure prediction method. I. Estimation procedure.

Proteins belonging to the TNC superfamily are known to be built of two, three, four, or six domains of closely similar amino acid sequences. Each domain binds no more than one calcium ion and shows a characteristic helix-loop-helix structure when in the calcium-bound state. Conformational properties of all the domains known so far have been analysed by us using a secondary structure prediction method (Garnier, J., Osguthorpe, D.J. & Robson, B. (1978). J. molec. Biol. 120, 97). Significant differences in distribution of residues predicted as being in the helical, beta-turn, and coil conformations have been found between the strongly, weakly, and non-binding domains. We could determine the ideal prediction pattern characteristic for the domains with the highest affinity for calcium. On the basis of our analysis and observations made by other authors we worked out a few simple rules which made it possible to compare conformational properties of a given domain with the ideal reference pattern and estimate, in this way, the Ca2+-binding constant of the domain. In native proteins the domains are known to be organized in pairs. The Ca2+-binding constant for a two-domain region could be evaluated from the sum of the estimation points attributed to each of its components. Using our method it is possible to predict the binding constants of typical domains and two-domain regins with a precision of one order of magnitude. Data on amino acid sequences and calcium-binding constants of all known proteins, believed to be the members of the TNC superfamily, have been reviewed. References to virtually all papers published on this subject before the end of 1987 are given.

Solution NMR structure and dynamics of human apo-S100A1 protein

S100A1 belongs to the EF-hand superfamily of calcium binding proteins. It is a representative of the S100 protein family based on amino acid sequence, three-dimensional structure, and biological function as a calcium signal transmitter. It is a homodimer of noncovalently bound subunits. S100A1, like most of other members of the S100 protein family, is a multifunctional, regulatory protein involved in a large variety of biological processes and closely associated with several human diseases. The three-dimensional structure of human apo-(i.e. calcium free)-S100A1 protein was determined by NMR spectroscopy (PDB 2L0P) and its backbone dynamics established by ¹⁵N magnetic relaxation. Comparison of these results with the structure and backbone dynamics previously determined for bovine apo-S100A1 protein modified by disulfide formation with β-mercaptoethanol at Cys 85 revealed that the secondary structure of both these proteins was almost identical, whereas the global structure of the latter was much more mobile than that of human apo-S100 protein. Differences between the structures of human and rat apo-S100A1 are also discussed.

Conformational study of two synthetic peptides with sequence analogies to the N-terminal fragment of RNase A.

The conformational properties of two synthetic model peptides, AEAAHAAEAAHMG (PA) and AEAAHAFEAAHMG (PF), have been studied using CD and 1H-NMR methods. In both peptides, glutamate and histidine residues are situated in such a way that two salt bridges between Glu- (i) and His+ (i + 3) can be formed. A salt bridge of this type (Glu- 9-His+ 12) was postulated previously to stabilize, to a great extent, the alpha-helical conformation of isolated N-terminal fragments of RNase A: C-peptide and S-peptide (A. Bierzyński, P.S. Kim and R.L. Baldwin, Proc. Natl. Acad. Sci. U.S.A. 79 (1982) 2470). Although in both PA and PF salt bridges between glutamates and histidines are formed, as demonstrated by the pH-titration curves of the glutamate gamma-proton signals, no traces of helical conformation have been detected. Evidently, the Glu- (i)-His+ (i + 3) salt bridges do not stabilize the alpha-helical conformation. A comparative analysis of PA and PF NMR spectra provides strong evidence that the phenylalanyl ring in PF interacts not only with the hydrophobic methyl groups of almost all alanine residues but also with the histidine rings and the glutamate side chains in their protonated as well as deprotonated forms. Similar interactions, involving Phe 8, can be expected in the N-terminal fragments of RNase and should be taken into account as an important factor determining the conformational properties of C- and S-peptides.