All cyclophilins and FK506 binding proteins are, individually and collectively, dispensable for viability in Saccharomyces cerevisiae

The cyclophilins and FK506 binding proteins (FKBPs) bind to cyclosporin A, FK506, and rapamycin and mediate their immunosuppressive and toxic effects, but the physiological functions of these proteins are largely unknown. Cyclophilins and FKBPs are ubiquitous and highly conserved enzymes that catalyze peptidyl-prolyl isomerization, a rate-limiting step during in vitro protein folding. We have addressed their functions by a genetic approach in the yeast Saccharomyces cerevisiae. Five cyclophilins and three FKBPs previously were identified in yeast. We identified four additional enzymes: Cpr6 and Cpr7, which are homologs of mammalian cyclophilin 40 that have also recently been independently isolated by others, Cpr8, a homolog of the secretory pathway cyclophilin Cpr4, and Fpr4, a homolog of the nucleolar FKBP, Fpr3. None of the eight cyclophilins or four FKBPs were essential. Surprisingly, yeast mutants lacking all 12 immunophilins were viable, and the phenotype of the dodecuplet mutant resulted from simple addition of the subtle phenotypes of each individual mutation. We conclude that cyclophilins and FKBPs do not play an essential general role in protein folding and find little evidence of functional overlap between the different enzymes. We propose that each cyclophilin and FKBP instead regulates a restricted number of unique partner proteins that remain to be identified.

Chaperonin filaments: The archaeal cytoskeleton?

Chaperonins are high molecular mass double-ring structures composed of 60-kDa protein subunits. In the hyperthermophilic archaeon Sulfolobus shibatae the two chaperonin proteins represent ≈4% of its total protein and have a combined intracellular concentration of >30 mg/ml. At concentrations ≥ 0.5 mg/ml purified chaperonins form filaments in the presence of Mg2+ and nucleotides. Filament formation requires nucleotide binding (not hydrolysis), and occurs at physiological temperatures in biologically relevant buffers, including a buffer made from cell extracts. These observations suggest that chaperonin filaments may exist in vivo and the estimated 4600 chaperonins per cell suggest that such filaments could form an extensive cytostructure. We observed filamentous structures in unfixed, uranyl-acetate-stained S. shibatae cells, which resemble the chaperonin filaments in size and appearance. ImmunoGold (Janssen) labeling using chaperonin antibodies indicated that many chaperonins are associated with insoluble cellular structures and these structures appear to be filamentous in some areas, although they could not be uranyl-acetate-stained. The existence of chaperonin filaments in vivo suggests a mechanism whereby their protein-folding activities can be regulated. More generally, the filaments themselves may play a cytoskeletal role in Archaea.

Hydrophobic sequence minimization of the α-lactalbumin molten globule

The molten globule, a widespread protein-folding intermediate, can attain a native-like backbone topology, even in the apparent absence of rigid side-chain packing. Nonetheless, mutagenesis studies suggest that molten globules are stabilized by some degree of side-chain packing among specific hydrophobic residues. Here we investigate the importance of hydrophobic side-chain diversity in determining the overall fold of the α-lactalbumin molten globule. We have replaced all of the hydrophobic amino acids in the sequence of the helical domain with a representative amino acid, leucine. Remarkably, the minimized molecule forms a molten globule that retains many structural features characteristic of a native α-lactalbumin fold. Thus, nonspecific hydrophobic interactions may be sufficient to determine the global fold of a protein.

Delayed Activation of the Store-operated Calcium Current Induced by Calreticulin Overexpression in RBL-1 Cells

Calreticulin (CRT) is a high-capacity, low-affinity Ca2+-binding protein located in the lumen of the endoplasmic reticulum (ER) of all eukaryotic cells investigated so far. Its high level of conservation among different species suggests that it serves functions fundamental to cell survival. The role originally proposed for CRT, i.e., the main Ca2+ buffer of the ER, has been obscured or even casted by its implication in processes as diverse as gene expression, protein folding, and cell adhesion. In this work we seek the role of CRT in Ca2+ storing and signaling by evaluating its effects on the kinetics and amplitude of the store-operated Ca2+ current (ICRAC). We show that, in the rat basophilic leukemia cell line RBL-1, overexpression of CRT, but not of its mutant lacking the high-capacity Ca2+-binding domain, markedly retards the ICRAC development, however, only when store depletion is slower than the rate of current activation. On the contrary, when store depletion is rapid and complete, overexpression of CRT has no effect. The present results are compatible with a major Ca2+-buffering role of CRT within the ER but exclude a direct, or indirect, role of this protein on the mechanism of ICRAC activation.

Tissue-specific Expression of Dominant Negative Mutant Drosophila HSC70 Causes Developmental Defects and Lethality

The Drosophila melanogaster HSC3 and HSC4 genes encode Hsc70 proteins homologous to the mammalian endoplasmic reticulum (ER) protein BiP and the cytoplasmic clathrin uncoating ATPase, respectively. These proteins possess ATP binding/hydrolysis activities that mediate their ability to aid in protein folding by coordinating the sequential binding and release of misfolded proteins. To investigate the roles of HSC3 (Hsc3p) and HSC4 (Hsc4p) proteins during development, GAL4-targeted gene expression was used to analyze the effects of producing dominant negatively acting Hsc3p (D231S, K97S) and Hsc4p (D206S, K71S) proteins, containing single amino acid substitutions in their ATP-binding domains, in specific tissues of Drosophila throughout development. We show that the production of each mutant protein results in lethality over a range of developmental stages, depending on the levels of protein produced and which tissues are targeted. We demonstrate that the functions of both Hsc3p and Hsc4p are required for proper tissue establishment and maintenance. Production of mutant Hsc4p, but not Hsc3p, results in induction of the stress-inducible Hsp70 at normal temperatures. Evidence is presented that lethality is caused by tissue-specific defects that result from a global accumulation of misfolded protein caused by lack of functional Hsc70. We show that both mutant Hsc3ps are defective in ATP-induced substrate release, although Hsc3p(D231S) does undergo an ATP-induced conformational change. We believe that the amino acid substitutions in Hsc3p interfere with the structural coupling of ATP binding to substrate release, and this defect is the basis for the mutant proteins’ dominant negative effects in vivo.

Functions of FKBP12 and Mitochondrial Cyclophilin Active Site Residues In Vitro and In Vivo in Saccharomyces cerevisiae

Cyclophilin and FK506 binding protein (FKBP) accelerate cis–trans peptidyl-prolyl isomerization and bind to and mediate the effects of the immunosuppressants cyclosporin A and FK506. The normal cellular functions of these proteins, however, are unknown. We altered the active sites of FKBP12 and mitochondrial cyclophilin from the yeast Saccharomyces cerevisiae by introducing mutations previously reported to inactivate these enzymes. Surprisingly, most of these mutant enzymes were biologically active in vivo. In accord with previous reports, all of the mutant enzymes had little or no detectable prolyl isomerase activity in the standard peptide substrate-chymotrypsin coupled in vitro assay. However, in a variation of this assay in which the protease is omitted, the mutant enzymes exhibited substantial levels of prolyl isomerase activity (5–20% of wild-type), revealing that these mutations confer sensitivity to protease digestion and that the classic in vitro assay for prolyl isomerase activity may be misleading. In addition, the mutant enzymes exhibited near wild-type activity with two protein substrates, dihydrofolate reductase and ribonuclease T1, whose folding is accelerated by prolyl isomerases. Thus, a number of cyclophilin and FKBP12 “active-site” mutants previously identified are largely active but protease sensitive, in accord with our findings that these mutants display wild-type functions in vivo. One mitochondrial cyclophilin mutant (R73A), and also the wild-type human FKBP12 enzyme, catalyze protein folding in vitro but lack biological activity in vivo in yeast. Our findings provide evidence that both prolyl isomerase activity and other structural features are linked to FKBP and cyclophilin in vivo functions and suggest caution in the use of these active-site mutations to study FKBP and cyclophilin functions.

Equilibrium constants and free energies in unfolding of proteins in urea solutions

A novel thermodynamic approach to the reversible unfolding of proteins in aqueous urea solutions has been developed based on the premise that urea ligands are bound cooperatively to the macromolecule. When successive stoichiometric binding constants have values larger than expected from statistical effects, an equation for moles of bound urea can be derived that contains imaginary terms. For a very steep unfolding curve, one can then show that the fraction of protein unfolded, B̄, depends on the square of the urea concentration, U, and is given by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}\bar {B}=\frac{{\mathit{A}}^{{\mathit{2}}}_{{\mathit{1}}}{\mathit{e}}^{{\mathrm{{\lambda}}}n\bar {B}}{\mathit{U}}^{{\mathit{2}}}}{{\mathrm{1\hspace{.167em}+\hspace{.167em}}}{\mathit{A}}^{{\mathrm{2}}}_{{\mathrm{1}}}{\mathit{e}}^{{\mathrm{{\lambda}}}\bar {B}}{\mathit{U}}^{{\mathrm{2}}}}{\mathrm{.}}\end{equation*}\end{document} A12 is the binding constant as B̄→ 0, and λ is a parameter that reflects the augmentation in affinities of protein for urea as the moles bound increases to the saturation number, n. This equation provides an analytic expression that reproduces the unfolding curve with good precision, suggests a simple linear graphical procedure for evaluating A12 and λ, and leads to the appropriate standard free energy changes. The calculated ΔG° values reflect the coupling of urea binding with unfolding of the protein. Some possible implications of this analysis to protein folding in vivo are described.

Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme

Recent advances in single molecule manipulation methods offer a novel approach to investigating the protein folding problem. These studies usually are done on molecules that are naturally organized as linear arrays of globular domains. To extend these techniques to study proteins that normally exist as monomers, we have developed a method of synthesizing polymers of protein molecules in the solid state. By introducing cysteines at locations where bacteriophage T4 lysozyme molecules contact each other in a crystal and taking advantage of the alignment provided by the lattice, we have obtained polymers of defined polarity up to 25 molecules long that retain enzymatic activity. These polymers then were manipulated mechanically by using a modified scanning force microscope to characterize the force-induced reversible unfolding of the individual lysozyme molecules. This approach should be general and adaptable to many other proteins with known crystal structures. For T4 lysozyme, the force required to unfold the monomers was 64 ± 16 pN at the pulling speed used. Refolding occurred within 1 sec of relaxation with an efficiency close to 100%. Analysis of the force versus extension curves suggests that the mechanical unfolding transition follows a two-state model. The unfolding forces determined in 1 M guanidine hydrochloride indicate that in these conditions the activation barrier for unfolding is reduced by 2 kcal/mol.

Site-specific conformational determination in thermal unfolding studies of helical peptides using vibrational circular dichroism with isotopic substitution

Understanding the detailed mechanism of protein folding requires dynamic, site-specific stereochemical information. The short time response of vibrational spectroscopies allows evaluation of the distribution of populations in rapid equilibrium as the peptide unfolds. Spectral shifts associated with isotopic labels along with local stereochemical sensitivity of vibrational circular dichroism (VCD) allow determination of the segment sequence of unfolding. For a series of alanine-rich peptides that form α-helices in aqueous solution, we used isotopic labeling and VCD to demonstrate that the α-helix noncooperatively unwinds from the ends with increasing temperature. For these blocked peptides, the C-terminal is frayed at 5°C. Ab initio level theoretical simulations of the IR and VCD band shapes are used to analyze the spectra and to confirm the conformation of the labeled components. The VCD signals associated with the labeled residues are amplified by coupling to the nonlabeled parts of the molecule. Thus small labeled segments are detectable and stereochemically defined in moderately large peptides in this report of site-specific peptide VCD conformational analysis.

Energetics of the interaction between water and the helical peptide group and its role in determining helix propensities

The alanine helix provides a model system for studying the energetics of interaction between water and the helical peptide group, a possible major factor in the energetics of protein folding. Helix formation is enthalpy-driven (−1.0 kcal/mol per residue). Experimental transfer data (vapor phase to aqueous) for amides give the enthalpy of interaction with water of the amide group as ≈−11.5 kcal/mol. The enthalpy of the helical peptide hydrogen bond, computed for the gas phase by quantum mechanics, is −4.9 kcal/mol. These numbers give an enthalpy deficit for helix formation of −7.6 kcal/mol. To study this problem, we calculate the electrostatic solvation free energy (ESF) of the peptide groups in the helical and β-strand conformations, by using the delphi program and parse parameter set. Experimental data show that the ESF values of amides are almost entirely enthalpic. Two key results are: in the β-strand conformation, the ESF value of an interior alanine peptide group is −7.9 kcal/mol, substantially less than that of N-methylacetamide (−12.2 kcal/mol), and the helical peptide group is solvated with an ESF of −2.5 kcal/mol. These results reduce the enthalpy deficit to −1.5 kcal/mol, and desolvation of peptide groups through partial burial in the random coil may account for the remainder. Mutant peptides in the helical conformation show ESF differences among nonpolar amino acids that are comparable to observed helix propensity differences, but the ESF differences in the random coil conformation still must be subtracted.

Dynamical principles in biological processes: A model of charge migration in proteins and DNA

The generalized master equations (GMEs) that contain multiple time scales have been derived quantum mechanically. The GME method has then been applied to a model of charge migration in proteins that invokes the hole hopping between local amino acid sites driven by the torsional motions of the floppy backbones. This model is then applied to analyze the experimental results for sequence-dependent long-range hole transport in DNA reported by Meggers et al. [Meggers, E., Michel-Beyerle, M. E., & Giese, B. (1998) J. Am. Chem. Soc. 120, 12950–12955]. The model has also been applied to analyze the experimental results of femtosecond dynamics of DNA-mediated electron transfer reported by Zewail and co-workers [Wan, C., Fiebig, T., Kelley, S. O., Treadway, C. R., Barton, J. K. & Zewail, A. H. (1999) Proc. Natl. Acad. Sci. USA 96, 6014–6019]. The initial events in the dynamics of protein folding have begun to attract attention. The GME obtained in this paper will be applicable to this problem.

Mutations in the DnaK chaperone affecting interaction with the DnaJ cochaperone

Hsp70 chaperones assist protein folding by ATP-controlled cycles of substrate binding and release. ATP hydrolysis is the rate-limiting step of the ATPase cycle that causes locking in of substrates into the substrate-binding cavity of Hsp70. This key step is strongly stimulated by DnaJ cochaperones. We show for the Escherichia coli Hsp70 homolog, DnaK, that stimulation by DnaJ requires the linked ATPase and substrate-binding domains of DnaK. Functional interaction with DnaJ is affected by mutations in an exposed channel located in the ATPase domain of DnaK. It is proposed that binding to this channel, possibly involving the J-domain, allows DnaJ to couple substrate binding with ATP hydrolysis by DnaK. Evolutionary conservation of the channel and the J-domain suggests conservation of the mechanism of action of DnaJ proteins.

Mode matches and their locations in the hydrophobic free energy sequences of peptide ligands and their receptor eigenfunctions

Patterns in sequences of amino acid hydrophobic free energies predict secondary structures in proteins. In protein folding, matches in hydrophobic free energy statistical wavelengths appear to contribute to selective aggregation of secondary structures in “hydrophobic zippers.” In a similar setting, the use of Fourier analysis to characterize the dominant statistical wavelengths of peptide ligands’ and receptor proteins’ hydrophobic modes to predict such matches has been limited by the aliasing and end effects of short peptide lengths, as well as the broad-band, mode multiplicity of many of their frequency (power) spectra. In addition, the sequence locations of the matching modes are lost in this transformation. We make new use of three techniques to address these difficulties: (i) eigenfunction construction from the linear decomposition of the lagged covariance matrices of the ligands and receptors as hydrophobic free energy sequences; (ii) maximum entropy, complex poles power spectra, which select the dominant modes of the hydrophobic free energy sequences or their eigenfunctions; and (iii) discrete, best bases, trigonometric wavelet transformations, which confirm the dominant spectral frequencies of the eigenfunctions and locate them as (absolute valued) moduli in the peptide or receptor sequence. The leading eigenfunction of the covariance matrix of a transmembrane receptor sequence locates the same transmembrane segments seen in n-block-averaged hydropathy plots while leaving the remaining hydrophobic modes unsmoothed and available for further analyses as secondary eigenfunctions. In these receptor eigenfunctions, we find a set of statistical wavelength matches between peptide ligands and their G-protein and tyrosine kinase coupled receptors, ranging across examples from 13.10 amino acids in acid fibroblast growth factor to 2.18 residues in corticotropin releasing factor. We find that the wavelet-located receptor modes in the extracellular loops are compatible with studies of receptor chimeric exchanges and point mutations. A nonbinding corticotropin-releasing factor receptor mutant is shown to have lost the signatory mode common to the normal receptor and its ligand. Hydrophobic free energy eigenfunctions and their transformations offer new quantitative physical homologies in database searches for peptide-receptor matches.

Rapid evolution of the DNA-binding site in LAGLIDADG homing endonucleases

Sequence analysis of chloroplast and mitochondrial large subunit rRNA genes from over 75 green algae disclosed 28 new group I intron-encoded proteins carrying a single LAGLIDADG motif. These putative homing endonucleases form four subfamilies of homologous enzymes, with the members of each subfamily being encoded by introns sharing the same insertion site. We showed that four divergent endonucleases from the I-CreI subfamily cleave the same DNA substrates. Mapping of the 66 amino acids that are conserved among the members of this subfamily on the 3-dimensional structure of I-CreI bound to its recognition sequence revealed that these residues participate in protein folding, homodimerization, DNA recognition and catalysis. Surprisingly, only seven of the 21 I-CreI amino acids interacting with DNA are conserved, suggesting that I-CreI and its homologs use different subsets of residues to recognize the same DNA sequence. Our sequence comparison of all 45 single-LAGLIDADG proteins identified so far suggests that these proteins share related structures and that there is a weak pressure in each subfamily to maintain identical protein–DNA contacts. The high sequence variability we observed in the DNA-binding site of homologous LAGLIDADG endonucleases provides insight into how these proteins evolve new DNA specificity.

CKAAPs DB: a conserved key amino acid positions database

The Conserved Key Amino Acid Positions DataBase (CKAAPs DB) provides access to an analysis of structurally similar proteins with dissimilar sequences where key residues within a common fold are identified. The derivation and significance of CKAAPs starting from pairwise structure alignments is described fully in Reddy et al. [Reddy,B.V.B., Li,W.W., Shindyalov,I.N. and Bourne,P.E. (2000) Proteins, in press]. The CKAAPs identified from this theoretical analysis are provided to experimentalists and theoreticians for potential use in protein engineering and modeling. It has been suggested that CKAAPs may be crucial features for protein folding, structural stability and function. Over 170 substructures, as defined by the Combinatorial Extension (CE) database, which are found in approximately 3000 representative polypeptide chains have been analyzed and are available in the CKAAPs DB. CKAAPs DB also provides CKAAPs of the representative set of proteins derived from the CE and FSSP databases. Thus the database contains over 5000 representative poly­peptide chains, covering all known structures in the PDB. A web interface to a relational database permits fast retrieval of structure-sequence alignments, CKAAPs and associated statistics. Users may query by PDB ID, protein name, function and Enzyme Classification number. Users may also submit protein alignments of their own to obtain CKAAPs. An interface to display CKAAPs on each structure from a web browser is also being implemented. CKAAPs DB is maintained by the San Diego Supercomputer Center and accessible at the URL http://ckaaps.sdsc.edu.