Cytochrome c′ folding triggered by electron transfer: Fast and slow formation of four-helix bundles

Reduced (FeII) Rhodopseudomonas palustris cytochrome c′ (Cyt c′) is more stable toward unfolding ([GuHCl]1/2 = 2.9(1) M) than the oxidized (FeIII) protein ([GuHCl]1/2 = 1.9(1) M). The difference in folding free energies (ΔΔGf° = 70 meV) is less than half of the difference in reduction potentials of the folded protein (100 mV vs. NHE) and a free heme in aqueous solution (≈−150 mV). The spectroscopic features of unfolded FeII–Cyt c′ indicate a low-spin heme that is axially coordinated to methionine sulfur (Met-15 or Met-25). Time-resolved absorption measurements after CO photodissociation from unfolded FeII(CO)–Cyt c′ confirm that methionine can bind to the ferroheme on the microsecond time scale [kobs = 5(2) × 104 s−1]. Protein folding was initiated by photoreduction (two-photon laser excitation of NADH) of unfolded FeIII–Cyt c′ ([GuHCl] = 2.02–2.54 M). Folding kinetics monitored by heme absorption span a wide time range and are highly heterogeneous; there are fast-folding (≈103 s−1), intermediate-folding (102–101 s−1), and slow-folding (10−1 s−1) populations, with the last two likely containing methionine-ligated (Met-15 or Met-25) ferrohemes. Kinetics after photoreduction of unfolded FeIII–Cyt c′ in the presence of CO are attributable to CO binding [1.4(6) × 103 s−1] and FeII(CO)–Cyt c′ folding [2.8(9) s−1] processes; stopped-flow triggered folding of FeIII–Cyt c′ (which does not contain a protein-derived sixth ligand) is adequately described by a single kinetics phase with an estimated folding time constant of ≈4 ms [ΔGf° = −33(3) kJ mol−1] at zero denaturant.

Multiple DNA and protein sequence alignment based on segment-to-segment comparison.

In this paper, a new way to think about, and to construct, pairwise as well as multiple alignments of DNA and protein sequences is proposed. Rather than forcing alignments to either align single residues or to introduce gaps by defining an alignment as a path running right from the source up to the sink in the associated dot-matrix diagram, we propose to consider alignments as consistent equivalence relations defined on the set of all positions occurring in all sequences under consideration. We also propose constructing alignments from whole segments exhibiting highly significant overall similarity rather than by aligning individual residues. Consequently, we present an alignment algorithm that (i) is based on segment-to-segment comparison instead of the commonly used residue-to-residue comparison and which (ii) avoids the well-known difficulties concerning the choice of appropriate gap penalties: gaps are not treated explicity, but remain as those parts of the sequences that do not belong to any of the aligned segments. Finally, we discuss the application of our algorithm to two test examples and compare it with commonly used alignment methods. As a first example, we aligned a set of 11 DNA sequences coding for functional helix-loop-helix proteins. Though the sequences show only low overall similarity, our program correctly aligned all of the 11 functional sites, which was a unique result among the methods tested. As a by-product, the reading frames of the sequences were identified. Next, we aligned a set of ribonuclease H proteins and compared our results with alignments produced by other programs as reported by McClure et al. [McClure, M. A., Vasi, T. K. & Fitch, W. M. (1994) Mol. Biol. Evol. 11, 571-592]. Our program was one of the best scoring programs. However, in contrast to other methods, our protein alignments are independent of user-defined parameters.

Protonatable residues at the cytoplasmic end of transmembrane helix-2 in the signal transducer HtrI control photochemistry and function of sensory rhodopsin I.

Neutral residue replacements were made of 21 acidic and basic residues within the N-terminal half of the Halobacterium salinarium signal transducer HtrI [the halobacterial transducer for sensory rhodopsin I (SRI)] by site-specific mutagenesis. The replacements are all within the region of HtrI that we previously concluded from deletion analysis to contain sites of interaction with the phototaxis receptor SRI. Immunoblotting shows plasmid expression of the htrI-sopI operon containing the mutations produces SRI and mutant HtrI in cells at near wild-type levels. Six of the HtrI mutations perturb photochemical kinetics of SRI and one reverses the phototaxis response. Substitution with neutral amino acids of Asp-86, Glu-87, and Glu-108 accelerate, and of Arg-70, Arg-84, and Arg-99 retard, the SRI photocycle. Opposite effects on photocycle rate cancel in double mutants containing one replaced acidic and one replaced basic residue. Laser flash spectroscopy shows the kinetic perturbations are due to alteration of the rate of reprotonation of the retinylidene Schiff base. All of these mutations permit normal attractant and repellent signaling. On the other hand, the substitution of Glu-56 with the isosteric glutamine converts the normally attractant effect of orange light to a repellent signal in vivo at neutral pH (inverted signaling). Low pH corrects the inversion due to Glu-56 -> Gln and the apparent pK of the inversion is increased when arginine is substituted at position 56. The results indicate that the cytoplasmic end of transmembrane helix-2 and the initial part of the cytoplasmic domain contain interaction sites with SRI. To explain these and previous results, we propose a model in which (i) the HtrI region identified here forms part of an electrostatic bonding network that extends through the SRI protein and includes its photoactive site; (ii) alteration of this network by photoisomerization-induced Schiff base deprotonation and reprotonation shifts HtrI between attractant and repellent conformations; and (iii) HtrI mutations and extracellular pH alter the equilibrium ratios of these conformations.

Template-nucleated alanine-lysine helices are stabilized by position-dependent interactions between the lysine side chain and the helix barrel.

The helicity in water has been determined for several series of alanine-rich peptides that contain single lysine residues and that are N-terminally linked to a helix-inducing and reporting template termed Ac-Hel1. The helix-propagating constant for alanine (sAla value) that best fits the properties of these peptides lies in the range of 1.01-1.02, close to the value reported by Scheraga and coworkers [Wojcik, J., Altmann, K.-H. & Scheraga, H.A. (1990) Biopolymers 30, 121-134], but significantly lower than the value assigned by Baldwin and coworkers [Chakrabartty, A., Kortemme, T. & Baldwin, R.L. (1994) Protein Sci. 3,843-852]. From a study of conjugates Ac-Hel1-Ala(n)-Lys-Ala(m)-NH2 and analogs in which the methylene portion of the lysine side chain is truncated, we find that the unusual helical stability of Ala(n)Lys peptides is controlled primarily by interactions of the lysine side chain with the helix barrel, and only passively by the alanine matrix. Using 1H NMR spectroscopy, we observe nuclear Overhauser effect crosspeaks consistent with proton-proton contacts expected for these interactions.

In vivo protein-DNA interactions at human DNA replication origin.

Protein-DNA interactions were studied in vivo at the region containing a human DNA replication origin, located at the 3' end of the lamin B2 gene and partially overlapping the promoter of another gene, located downstream. DNase I treatment of nuclei isolated from both exponentially growing and nonproliferating HL-60 cells showed that this region has an altered, highly accessible, chromatin structure. High-resolution analysis of protein-DNA interactions in a 600-bp area encompassing the origin was carried out by the in vivo footprinting technique based on the ligation-mediated polymerase chain reaction. In growing HL-60 cells, footprints at sequences homologous to binding sites for known transcription factors (members of the basic-helix-loop-helix family, nuclear respiratory factor 1, transcription factor Sp1, and upstream binding factor) were detected in the region corresponding to the promoter of the downstream gene. Upon conversion of cells to a nonproliferative state, a reduction in the intensity of these footprints was observed that paralleled the diminished transcriptional activity of the genomic area. In addition to these protections, in close correspondence to the replication initiation site, a prominent footprint was detected that extended over 70 nucleotides on one strand only. This footprint was absent from nonproliferating HL-60 cells, indicating that this specific protein-DNA interaction might be involved in the process of origin activation.

Thermodynamic parameters for the helix-coil transition of oligopeptides: molecular dynamics simulation with the peptide growth method.

The helix-coil transition equilibrium of polypeptides in aqueous solution was studied by molecular dynamics simulation. The peptide growth simulation method was introduced to generate dynamic models of polypeptide chains in a statistical (random) coil or an alpha-helical conformation. The key element of this method is to build up a polypeptide chain during the course of a molecular transformation simulation, successively adding whole amino acid residues to the chain in a predefined conformation state (e.g., alpha-helical or statistical coil). Thus, oligopeptides of the same length and composition, but having different conformations, can be incrementally grown from a common precursor, and their relative conformational free energies can be calculated as the difference between the free energies for growing the individual peptides. This affords a straightforward calculation of the Zimm-Bragg sigma and s parameters for helix initiation and helix growth. The calculated sigma and s parameters for the polyalanine alpha-helix are in reasonable agreement with the experimental measurements. The peptide growth simulation method is an effective way to study quantitatively the thermodynamics of local protein folding.

Mxi2, a mitogen-activated protein kinase that recognizes and phosphorylates Max protein.

We describe Mxi2, a human protein that interacts with Max protein, the heterodimeric partner of the Myc oncoprotein. Mxi2 encodes a 297-residue protein whose sequence indicates that it is related to extracellular signal-regulated kinases (ERK protein kinases). Mxi2 in yeast interacts with Max and with the C terminus of c-Myc. Mxi2 phosphorylates Max both in vitro and in vivo. The Mxi2 putative substrate recognition region has sequence similarity to the helix-loop-helix region in Max and c-Myc, suggesting that substrate recognition might be mediated via this motif. Phosphorylation by Mxi2 may affect the ability of Max to oligomerize with itself and its partners, bind DNA, or regulate gene expression.

Alternate protein frameworks for molecular recognition.

In an effort to determine whether proteins with structures other than the immunoglobulin fold can be used to mimic the ligand binding properties of antibodies, we generated a library from the four-helix bundle protein cytochrome b562 in which the two loops were randomized. Panning of this library against the bovine serum albumin (BSA) conjugate of N-methyl-p-nitrobenzylamine derivative 1 by phage display methods yielded cytochromes in which residues Trp-20, Arg-21, and Ser-22 in loop A and Arg-83 and Trp-84 in loop B were conserved. The individual mutants, which fold into native-like structure, bind selectively to the BSA-1 conjugate with micromolar dissociation constants (Kd), in comparison to a monoclonal antibody that binds selectively to 1 with a Kd of 290 nM. These and other antibody-like receptors may prove useful as therapeutic agents or as reagents for both intra- and extracellular studies.

Heat shock protein hsp90 regulates dioxin receptor function in vivo.

The dioxin (aryl hydrocarbon) receptor is a ligand-dependent basic helix-loop-helix (bHLH) factor that binds to xenobiotic response elements of target promoters upon heterodimerization with the bHLH partner factor Arnt. Here we have replaced the bHLH motif of the dioxin receptor with a heterologous DNA-binding domain to create fusion proteins that mediate ligand-dependent transcriptional enhancement in yeast (Saccharomyces cerevisiae). Previously, our experiments indicated that the ligand-free dioxin receptor is stably associated with the 90-kDa heat shock protein, hsp90. To investigate the role of hsp90 in dioxin signaling we have studied receptor function in a yeast strain where hsp90 expression can be down-regulated to about 5% relative to wild-type levels. At low levels of hsp90, ligand-dependent activation of the chimeric dioxin receptor construct was almost completely inhibited, whereas the activity of a similar chimeric construct containing the structurally related Arnt factor was not affected. Moreover, a chimeric dioxin receptor construct lacking the central ligand- and hsp90-binding region of the receptor showed constitutive transcriptional activity in yeast that was not impaired upon down-regulation of hsp90 expression levels. Thus, these data suggest that hsp90 is a critical determinant of conditional regulation of dioxin receptor function in vivo via the ligand-binding domain.

On the distribution of amino acid residues in transmembrane alpha-helix bundles.

The periodic distribution of residues in the sequence of 469 putative transmembrane alpha-helices from eukaryotic plasma membrane polytopic proteins has been analyzed with correlation matrices. The method does not involve any a priori assumption about the secondary structure of the segments or about the physicochemical properties of individual amino acid residues. Maximal correlation is observed at 3.6 residues per period, characteristic of alpha-helices. A scale extracted from the data describes the propensity of the various residues to lie on the same or on opposite helix faces. The most polar face of transmembrane helices, presumably that buried in the protein core, shows a strong enrichment in aromatic residues, while residues likely to face the fatty acyl chains of lipids are largely aliphatic.

Expression and biochemical analysis of the entire HIV-2 gp41 ectodomain: determinants of stability map to N- and C-terminal sequences outside the 6-helix bundle core

The folding of HIV gp41 into a 6-helix bundle drives virus-cell membrane fusion. To examine the structural relationship between the 6-helix bundle core domain and other regions of gp41, we expressed in Escherichia coli, the entire ectodomain of HIV-2(ST) gp41 as a soluble, trimeric maltose-binding protein (MBP)/gp41 chimera. Limiting proteolysis indicated that the Cys-591-Cys-597 disulfide-bonded region is outside a core domain comprising two peptides, Thr-529-Trp-589 and Val-604-Ser-666. A biochemical examination of MBP/gp41 chimeras encompassing these core peptides; indicated that the N-terminal polar segment, 521-528, and C-terminal membrane-proximal segment, 658-666, cooperate in stabilizing the ectodomain. A functional interaction between sequences outside the gp41 core may contribute energy to membrane fusion. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Metal Clips Induce Folding of a Short Unstructured Peptide into an a-Helix via Turn Conformations in Water. Kinetic versus Thermodynamic Products

Short peptides corresponding to two to four a-helical turns of proteins are not thermodynamically stable helices in water. Unstructured octapeptide Ac-His1*-Ala2-Ala3-His4*-His5*-Glu6-Leu7-His8*-NH2 (1) reacts with two [Pd ((NH2)-N-15(CH2)(2) (NH2)-N-15)(NO3)(2)] in water to form a kinetically stable intermediate, [{Pden}(2)-{(1,4)(5,8)-peptide}](2), in which two 19-membered metallocyclic rings stabilize two peptide turns. Slow subsequent folding to a thermodynamically more stable two-turn a-helix drives the equilibrium to [{Pden}(2)-{(1,5)(4,8)-peptide}] (3), featuring two 22-membered rings. This transformation from unstructured peptide via turns to an a-helix suggests that metal clips might be useful probes for investigating peptide folding.

Mutational analysis of the large periplasmic loop 7-8 of the putrescine transporter PotE in Escherichia coli

The PotE protein is a putrescine-ornithine antiporter found in many gram-negative bacteria. It is a member of the APA family of transporters and has 12 predicted alpha-helical transmembrane spanning segments (TMS). While the substrate binding site has previously been mapped to a region near the surface of the cytoplasmic lipid layer, no structural feature within the periplasmic domains of PotE have been shown to be important for function. We examined the role of the only large outer loop, situated between transmembrane spanning segment 7 and 8, in putrescine uptake. Deletion of the highly conserved amino acids in the region closest to transmembrane spanning segment 7 produced a protein with little activity. Glycine-scanning mutagenesis of this region showed that Val(249) and Leu(254) were required for optimal transporter function. The V249G mutant transported putrescine at a lower maximal rate compared to wild-type (WT) but with the same substrate binding affinity. In contrast, the L254G mutant had a higher substrate affinity. A series of Val(249) mutants indicated that the hydrophobicity of this residue, which is located at or near the membrane surface, is important for PotE function. Secondary structure predictions of the large outer loop indicated the presence of a hydrophobic alpha-helix in the centre with a hydrophobic region at each end suggesting that the loop was not entirely exposed to the aqueous periplasmic space. The study shows that loop 7-8 is important for PotE function, possibly by forming a re-entrant loop in the channel of the transporter. (C) 2003 Elsevier Ltd. All rights reserved.

Structure and dynamics of ASC2, a pyrin domain-only protein that regulates inflammatory signaling

Pyrin domain (PYD)-containing proteins are key components of pathways that regulate inflammation, apoptosis, and cytokine processing. Their importance is further evidenced by the consequences of mutations in these proteins that give rise to autoimmune and hyperinflammatory syndromes. PYDs, like other members of the death domain ( DD) superfamily, are postulated to mediate homotypic interactions that assemble and regulate the activity of signaling complexes. However, PYDs are presently the least well characterized of all four DD subfamilies. Here we report the three-dimensional structure and dynamic properties of ASC2, a PYD-only protein that functions as a modulator of multidomain PYD-containing proteins involved in NF-KB and caspase-1 activation. ASC2 adopts a six-helix bundle structure with a prominent loop, comprising 13 amino acid residues, between helices two and three. This loop represents a divergent feature of PYDs from other domains with the DD fold. Detailed analysis of backbone N-15 NMR relaxation data using both the Lipari-Szabo model-free and reduced spectral density function formalisms revealed no evidence of contiguous stretches of polypeptide chain with dramatically increased internal motion, except at the extreme N and C termini. Some mobility in the fast, picosecond to nanosecond timescale, was seen in helix 3 and the preceding alpha 2-alpha 3 loop, in stark contrast to the complete disorder seen in the corresponding region of the NALP1 PYD. Our results suggest that extensive conformational flexibility in helix 3 and the alpha 2-alpha 3 loop is not a general feature of pyrin domains. Further, a transition from complete disorder to order of the alpha 2-alpha 3 loop upon binding, as suggested for NALP1, is unlikely to be a common attribute of pyrin domain interactions.

Helix Explorer : une nouvelle base de données de structures de protéines

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.