The role of context on alpha-helix stabilization: host-guest analysis in a mixed background peptide model.

The helix content of a series of peptides containing single substitutions of the 20 natural amino acids in a new designed host sequence, succinyl-YSEEEEKAKKAXAEEAEKKKK-NH2, has been determined using CD spectroscopy. This host is related to one previously studied, in which triple amino acid substitutions were introduced into a background of Glu-Lys blocks completely lacking alanine. The resulting free energies show that only Ala and Glu- prove to be helix stabilizing, while all other side chains are neutral or destabilizing. This agrees with results from studies of alanine-rich peptide modela, but not the previous Glu-Lys block oligomers in which Leu and Met also stabilize helix. The helix propensity scale derived from the previous block oligomers correlated well with the frequencies of occurrence of different side chains in helical sequences of proteins, whereas the values from the present series do not. The role of context in determining scales of helix propensity values is discussed, and the ability of algorithms designed to predict helix structure from sequence is compared.     

A Highly Unusual Thioester Bond in a Pilus Adhesin Is Required for Efficient Host Cell Interaction

Many bacterial pathogens present adhesins at the tips of long macromolecular filaments known as pili that are often important virulence determinants. Very little is known about how pili presented by Gram-positive pathogens mediate host cell binding. The crystal structure of a pilus adhesin from the important human pathogen Streptococcus pyogenes reveals an internal thioester bond formed between the side chains of a cysteine and a glutamine residue. The presence of the thioester was verified using UV-visible spectroscopy and mass spectrometry. This unusual bond has only previously been observed in thioester domains of complement and complement-like proteins where it is used to form covalent attachment to target molecules. The structure also reveals two intramolecular isopeptide bonds, one of these formed through a Lys/Asp residue pair, which are strategically positioned to confer protein stability. Removal of the internal thioester by allele-replacement mutagenesis in S. pyogenes severely compromises bacterial adhesion to model host cells. Although current paradigms of bacterial/host cell interaction envisage strong non-covalent interactions, the present study suggests cell adhesion could also involve covalent bonds.     

Reduction of interchain disulfide bonds precedes the dislocation of Ig-mu chains from the endoplasmic reticulum to the cytosol for proteasomal degradation

Proteins that fail to fold or assemble in the endoplasmic reticulum (ER) are generally dislocated across the membrane to be degraded by cytosolic proteasomes. To investigate how the quality control machinery handles individual subunits that are part of covalent oligomers, we have analyzed the fate of transport-competent Ig light (L) chains that form disulfide bonds with short-lived mu heavy chains. When expressed alone, L chains are secreted. In cells producing excess mu, most L chains are retained in the ER as covalent mu-L or mu2-L2 complexes. While mu chains present in these complexes are degraded by proteasomes, L chains are stable. Few L chains are secreted; most reassociate with newly synthesized mu chains. Therefore, interchain disulfide bonds are reduced in the ER lumen before the dislocation of mu chains in a site from which freed L chains can be rapidly reinserted in the assembly line. The ER can thus sustain the simultaneous formation and reduction of disulfide bonds.     

Nucleotide sequence of nuclear 5.7S RNA of mouse cells

Viable tumor cells were examined by ¹⁹F-NMR spectroscopy after treatment with 4-trifluoromethyl 2,6-dinitrophenyl sulphonate (CF₃-DNBS), which is an analog of 2,4,6-trinitrophenyl sulphonate (TNBS). The presence of a strong ¹⁹F-NMR signal from treated cells suggested the binding of the “probe”. Treatment of labelled cells with proteolytic enzymes significantly decreased the signals, suggesting that the label was essentially bound to the cell surface macromolecules. A proportion of the material bound to the cell was removable by dialysis of cell extracts against a structural analog, suggesting that some CF₃-DNBS-cell membrane bonds were not covalent. The existence of such non-covalent bonds has also been confirmed with soluble proteins. By the same approach, it was also found that ¹⁴C-TNBS formed covalent and non-covalent bonds with tumor cells.     

Unimolecular and bimolecular oxidoreduction reactions involving diprotein complexes of cytochrome c and plastocyanin. Dependence of electron-transfer reactivity on charge and orientation of the docked metalloproteins.

Cytochrome c and plastocyanin form an electrostatic complex, which can be reinforced by amide bonds in the presence of a carbodiimide. Besides this cross-linking, carbodiimide also converts carboxylate side chains into neutral N-acylurea groups. Four derivatives of the covalent diprotein complex, which differ in the degree of this charge neutralization, are separated by cation-exchange chromatography. Electron-transfer reactions at different ionic strengths involving the electrostatic complex and the four derivatives of the covalent complex are studied by laser flash photolysis with flavin semiquinones as reducing agents. The reactivity of the associated proteins toward external reductants cannot be predicted simply on the basis of this reactivity of the separate proteins. Qualitative analysis of the dependence on ionic strength of the reactions between FMN semiquinone and the covalent derivatives indicates sites at which this reductant interacts with the cross-linked proteins. The surprisingly small steric shielding of the protein redox sites in the covalent complex, as deduced from the reactions at high ionic strength, may indicate that the proteins have multiple reaction domains on their surfaces or that the complex is dynamical or both. The intracomplex (unimolecular) electron-transfer reaction is fast in the electrostatic complex (ket = 1300 +/- 200 s-1) but undetectably slow in each of the four derivatives of the covalent complex (ket less than 0.2 s-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Specificity of alpha 2-macroglobulin covalent cross-linking for the active domain of proteinases

The reaction of alpha 2-macroglobulin (alpha 2M) with the two-chain enzyme plasma kallikrein results in covalent bond formation between the catalytic subunit and the inhibitor. We have recently published a model of alpha 2M which suggests that this phenomenon may be a general mechanism when multisubunit proteinases are inactivated by alpha 2M. In order to test this hypothesis, we studied the reactions of factor Xa, plasmin, streptokinase-plasmin and alpha-thrombin with alpha 2M. In the case of factor Xa the catalytic heavy chain demonstrated greater than 99% covalent incorporation while over 97% of the light chain failed to crosslink to the inhibitor. Preferential binding of the catalytic light chains of plasmin (70% covalent incorporation) and plasmin in complex with streptokinase (79% covalent incorporation) was also observed. Finally, 82% covalent incorporation of the catalytic heavy chain of alpha-thrombin was found. These studies demonstrate that in the case of multisubunit proteinases, the chain containing the active site demonstrates preferential binding as predicted by the model supporting placement of the site of covalent binding close to the "bait region" of alpha 2M.     

C-type cytochromes: diverse structures and biogenesis systems pose evolutionary problems

C-type cytochromes are a structurally diverse group of haemoproteins, which are related by the occurrence of haem covalently attached to a polypeptide via two thioether bonds formed by the vinyl groups of haem and cysteine side chains in a CXXCH peptide motif. Remarkably, three different post-translational systems for forming these cytochromes have been identified. The evolution of both the proteins themselves and the biogenesis systems poses many questions to which answers are currently being sought. In this article we review the progress that has been made in understanding the need for covalent attachment of haem to proteins in cytochromes c and the complex systems involved in their formation.     

Cleavage C-terminal to Asp leads to covalent crosslinking of long-lived human proteins

With age, long-lived proteins in the human body deteriorate, which can have consequences both for aging and disease. The aging process is often associated with the formation of covalently crosslinked proteins. Currently our knowledge of the mechanism of formation of these crosslinks is limited. In this study, proteomics was used to characterize sites of covalent protein-protein crosslinking and identify a novel mechanism of protein-protein crosslinking in the adult human lens. In this mechanism, Lys residues are crosslinked to C-terminal Asp residues that are formed by non-enzymatic protein truncation. Ten different crosslinks were identified in major lens proteins such as αA-crystallin, αB-crystallin and AQP0. Crosslinking in AQP0 increased significantly with age and also increased significantly in cataract lenses compared with normal lenses. Using model peptides, a mechanism of formation of the Lys-Asp crosslink was elucidated. The mechanism involves spontaneous peptide cleavage on the C-terminal side of Asp residues which can take place in the pH range 5–7.4. Cleavage appears to involve attack by the side chain carboxyl group on the adjacent peptide bond, resulting in the formation of a C-terminal Asp anhydride. This anhydride intermediate can then either react with water to form Asp, or with a nucleophile, such as a free amine group to form a crosslink. If an ε-amino group of Lys or an N-terminal amine group attacks the anhydride, a covalent protein-protein crosslink will be formed. This bi-phasic mechanism represents the first report to link two spontaneous events: protein cleavage and crosslinking that are characteristic of long-lived proteins.     

Co-translational modification of nascent immunoglobulin heavy and light chains

We have investigated the in vivo co-translational covalent modification of nascent immunoglobulin heavy and light chains. Nascent polypeptides were separated from completed polypeptides by ion-exchange chromatography of solubilized ribosomes on QAE-Sephadex. First, we have demonstrated that MPC 11 nascent heavy chains are quantitatively glycosylated very soon after the asparaginyl acceptor site passes through the membrane into the cisterna of the rough endoplasmic reticulum. Nonglycosylated completed heavy chains of various classes cannot be glycosylated after release from the ribosome, due either to rapid intramolecular folding and/or intermolecular assembly, which cause the acceptor site to become unavailable for the glycosylation enzyme. Second, we have shown that the formation of the correct intrachain disulfide loop within the first light chain domain occurs rapidly and quantitatively as soon as the appropriate cysteine residues of the nascent light chain pass through the membrane into the cisterna of the endoplasmic reticulum. The intrachain disulfide loop in the second or constant region domain of the light chain is not formed on nascent chains, because one of the cysteine residues involved in this disulfide bond does not pass through the endoplasmic reticulum membrane prior to chain completion and release from the ribosome. Third, we have demonstrated that some of the initial covalent assembly (formation of interchain disulfide bonds) occurs on nascent heavy chains prior to their release from the ribosome. The results are consistent with the pathway of covalent assembly of the cell line, in that completed light chains are assembled onto nascent heavy chains in MPC 11 cells (IgG₂b), where a heavy-light half molecule is the major initial covalent intermediate; and completed heavy chains are assembled onto nascent heavy chains in MOPC 21 cells (IgG₁), where a heavy chain dimer is the major initial disulfide linked intermediate.     

Rheological characterization and dissolution kinetics of fibrin gels crosslinked by a microbial transglutaminase

Various fibrin gels were prepared with a microbial transglutaminase under miscellaneous conditions. The gels were characterized through their rheological properties. The influence of fibronectin addition and that of covalent bonding on the viscoelastic characteristics were evaluated. Gel elasticity is proportional to fibrinogen concentration but shows a nonlinear dependence on transglutaminase concentration. Additional crosslink of fibronectin in fibrin gels has no effect on the rheological character of the matrix. Dissolution kinetics in concentrated urea solutions evidences the role of covalent bonds on gel stability. The rheological properties and gel stability are discussed in relation with the enzyme-catalyzed covalent bonding. The microbial enzyme reactions are compared to those of FXIII and tissue transglutaminases.     

Thermostability of α-mannosidase in plasma from cystic fibrosis patients and carriers

The hypothesis presented is that the different classes of c-type cytochrome originated as proteins located in the bacterial periplasmic space, or on the periplasmic side of the cytoplasmic membrane. In these locations, covalent bonds between haem and protein prevented the haem from being lost to the surrounding medium. Subsequent evolution has led to internal location of c-type cytochromes in eucaryotes and cyanobacteria. The covalent links have been retained because of their structural role; a b-type cytochrome could be created with similar molecular properties, but its formation would require a large evolutionary jump. If this hypothesis is correct, it should be useful in unravelling electron transport chains with unconventional donors or acceptors. Apparent exceptions deserve further investigation.     

Anticooperativity in a Glu-Lys-Glu salt bridge triplet in an isolated alpha-helical peptide

Salt bridges between oppositely charged side chains are well-known to stabilize protein structure, though their contributions vary considerably. Here we study Glu-Lys and Lys-Glu salt bridges, formed when the residues are spaced i, i + 4 surface of an isolated alpha-helix in aqueous solution. Both are stabilizing by -0.60 and -1.02 kcal/mol, respectively, when the interacting residues are fully charged. When the side chains are spaced i, i + 4, i + 8, forming a Glu-Lys-Glu triplet, the second salt bridge provides no additional stabilization to the helix. We attribute this to the inability of the central Lys to form two salt bridges simultaneously. Analysis of these salt bridges in protein structures shows that the Lys-Glu interaction is dominant, with the side chains of the Glu-Lys pair far apart.     

Involvement of calponin and caldesmon in sustained contraction of arterial smooth muscle.

The molecular mechanism of smooth muscle contraction was approached by a novel method, covalent 14C-labeling. Intra- and intermolecular protein interactions during contractile activity are reflected by changed reactivity of protein side chains; these can be detected by reagents which readily permeate through the muscle membrane without affecting the contractility and form covalent bonds with proteins in the muscle. The incorporation of 14CH2ICONH2 into proteins of 1-hour histamine contracted versus resting porcine carotid arterial muscles was determined. Out of fourteen 14C-labeled proteins analyzed, only two showed a change in reactivity during sustained contraction. The incorporation of 14CH2ICONH2 into calponin and caldesmon in contracted muscles was about 66% of that into these same proteins in resting muscles. A transformation of calponin and caldesmon molecules from an extended to a more compact conformation explains the decreased reactivity.     

Cytochrome c biogenesis System I

Cytochromes c are widespread respiratory proteins characterized by the covalent attachment of heme. The formation of c-type cytochromes requires, in all but a few exceptional cases, the formation of two thioether bonds between the two cysteine sulfurs in a -CXXCH- motif in the protein and the vinyl groups of heme. The vinyl groups of the heme are not particularly activated and therefore the addition reaction does not physiologically occur spontaneously in cells. There are several diverse post-translational modification systems for forming these bonds. Here, we describe the complex multiprotein cytochrome c maturation (Ccm) system (in Escherichia coli comprising the proteins CcmABCDEFGH), also called System I, that performs the heme attachment. System I is found in plant mitochondria, archaea and many Gram-negative bacteria; the systems found in other organisms and organelles are described elsewhere in this minireview series.     

Protein acylation in Tetrahymena

Examination of exhaustively delipidated Tetrahymena mimbres cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of several protein bands containing covalently linked fatty acids. Palmitic (16:0) and stearic (18:0) acids together accounted for approximately 90% of the protein-linked acyl chains, with myristic acid (14:0) comprising most of the remainder. Each of these three fatty acids was present mainly in alkali-stable linkage, indicating that unlike most other systems examined, fatty acids are attached to proteins of Tetrahymena principally by amide bonds. Smaller proportions of the acyl chains were susceptible to release by hydroxylaminolysis or by alkaline hydrolysis as would be expected from an ester linkage. The protein-bound acyl chains accounted for 0.3% of the cells' total fatty acids. They closely resembled in composition the highly saturated free fatty acid pool but not the vast pool of glycerolipid-associated fatty acids, which were mainly unsaturated. Cells subjected to thermal stress by rapid chilling from 39 to 15 degrees C responded by sharply increasing the ratio of palmitate to stearate in covalent association with proteins.     

Contemporary strategies for the stabilization of peptides in the alpha-helical conformation

Herein we review contemporary synthetic and protein design strategies to stabilize the alpha-helical motif in short peptides and miniature proteins. Advances in organometallic catalyst design, specifically for the olefin metathesis reaction, enable the use of hydrocarbon bridges to either crosslink side chains of specific residues or mimic intramolecular hydrogen bonds with carbon-carbon bonds. The resulting hydrocarbon-stapled and hydrogen bond surrogate alpha-helices provide unique synthetic ligands for targeting biomolecules. In the protein design realm, several classes of miniature proteins that display stable helical domains have been engineered and manipulated with powerful in vitro selection technologies to yield libraries of sequences that retain their helical folds. Rational re-design of these scaffolds provide distinctive reagents for the modulation of protein-protein interactions.     

Role of heme-protein covalent bonds in mammalian peroxidases. Protection of the heme by a single engineered heme-protein link in horseradish peroxidase

Oxidation of SCN-, Br-, and Cl- (X-) by horseradish peroxidase (HRP) and other plant and fungal peroxidases results in the addition of HOX to the heme vinyl group. This reaction is not observed with lactoperoxidase (LPO), in which the heme is covalently bound to the protein via two ester bonds between carboxylic side chains and heme methyl groups. To test the hypothesis that the heme of LPO and other mammalian peroxidases is protected from vinyl group modification by the hemeprotein covalent bonds, we prepared the F41E mutant of HRP in which the heme is attached to the protein via a covalent bond between Glu41 and the heme 3-methyl. We also examined the E375D mutant of LPO in which only one of the two normal covalent heme links is retained. The prosthetic heme groups of F41E HRP and E375D LPO are essentially not modified by the HOBr produced by these enzymes. The double E375D/D225E mutant of LPO that can form no covalent bonds is inactive and could not be examined. These results unambiguously demonstrate that a single heme-protein link is sufficient to protect the heme from vinyl group modification even in a protein (HRP) that is normally highly susceptible to this reaction. The results directly establish that one function of the covalent heme-protein bonds in mammalian peroxidases is to protect their prosthetic group from their highly reactive metabolic products.     

Antiparallel alignment of the two protomers of the regulatory subunit dimer of cAMP-dependent protein kinase I

The purified type I regulatory subunit of cAMP-dependent protein kinase is a dimeric protein, and the two protomers of the dimer are linked by two interchain disulfide bonds. The disulfide linkages that join these two polypeptide chains have been identified in order to provide a structural basis for the orientation of the two chains in the asymmetric dimer. Disulfide bonds were found to exist exclusively between Cys-16 and Cys-37, and this assignment, thus, establishes a general antiparallel alignment of the two chains. Two other homologous proteins, the type II regulatory subunit and the cGMP-dependent protein kinase also are dimeric proteins. In all three proteins, a relatively small, nonhomologous, amino-terminal segment of the polypeptide chain is essential for maintaining the dimeric aggregation state.     

Laser-Raman and infrared spectroscopic studies of protein conformation in the eggshell of the fish Salmo gairdneri

Laser-Raman and infrared spectroscopic studies reveal abundant beta-pleated sheet conformation in the eggshell proteins of the fish Salmo gairdneri. This secondary structure is the underlying molecular conformation, dictating the formation of the helicoidal architecture of the eggshell. Disulphide bonds crosslink the eggshell proteins of the fertilized eggs and are apparently found in g-g-g (gauche-gauche-gauche), g-g-t (gauche-gauche-trans) and t-g-t (trans-gauche-trans) conformation. There is no evidence for the existence of free sulphydryls. The tyrosines appear to act as hydrogen-bond acceptors, whereas the aromatic residues phenylalanine and tryptophan are also eggshell protein constituents.     

A nuclear matrix protein binds very tightly to DNA in the avian beta-globin gene enhancer

Current evidence suggests that DNA is covalently attached to proteins in the nuclear matrix of eukaryotic cells and that specific DNA sequences are tightly associated with the nuclear matrix. However, it has not been documented that specific DNA sequences can become covalently attached to nuclear matrix protein. We have examined the binding of cloned DNA sequences that contain the avian beta-globin gene enhancer, a region previously shown to be matrix associated in erythroid cells in vivo, with nuclear matrices from several avian tissue sources to determine if covalent DNA-protein bonds are formed. Our results indicate that sequence-specific DNA-protein complexes that are resistant to denaturation by SDS, boiling, and phenol and disulfide reduction are formed. Excess protein, capable of forming very tight bonds with DNA that contains the beta-globin gene enhancer, is present in cells in which matrix attachment of this DNA sequence is not detected in vivo. Evidence is presented that suggests that the protein to which DNA forms very tight bonds is not topoisomerase II. These results are discussed in relation to current models of the nuclear matrix and the utility of in vitro assays of matrix attachment regions using cloned DNA.