Conformational aspects of N-glycosylation of proteins. Studies with linear and cyclic peptides as probes

Conformational aspects of N-glycosylation of glycoproteins have been studied by using a series of peptides which contained, in addition to the `marker sequence' Asn-Gly-Thr, two cysteine residues in various positions of the peptide chain. The presence of two cysteines permitted a partial fixation of the above triplet sequence in cyclic structures of various size by intramolecular disulphide bond formation. Comparison of the glycosyl acceptor properties of the linear peptides and their corresponding cyclic analogues allows the following statements. The considerably lower acceptor capabilities of the cyclic derivatives indicate that the restriction of rotational degrees of freedom imposed by disulphide bonding results in a conformation which hinders a favourable interaction of the peptide substrate with the N-glycosyltransferase. On the other hand, the glycosylation rate of linear peptides increases with increasing chain length, suggesting that the amino acids on both the N- and C-terminal side of the `marker sequence' may contribute to a considerable extent to the induction of an `active' conformation. Realization of a potential sugar attachment site requires a hydrogen bond interaction within the `marker sequence' between the oxygen of threonine (serine) as the hydrogen bond acceptor and the β-amide of asparagine as the donor [Bause & Legler (1981) Biochem. J. 195, 639–644]. This interaction is obviously facilitated when the peptide chain can adopt a conformation which resembles a β-turn or other loop structure. The available experimental and statistical data are discussed in terms of possible structural features for N-glycosylation, with the aid of space-filling models.     

Synthetic peptides as probes for conformational preferences of domains of membrane receptors

Peptide models have been widely used to investigate conformational aspects of domains of proteins since the early 1950s. A pioneer in this field was Dr. Murray Goodman, who applied a battery of methodologies to study the onset of structure in homooligopeptides. This article reviews some of Dr. Goodman's contributions, and reports recent studies using linear and constrained peptides corresponding to the first extracellular loop and linear peptides corresponding to the sixth transmembrane domain of a G-protein coupled receptor from the yeast Saccharomyces cerevisiae. Peptides containing 30-40 residues were synthesized using solid-phase methods and purified to near homogeneity by reversed phase high performance liquid chromatography. CD and NMR analyses indicated that the first extracellular loop peptides were mostly flexible in water, and assumed some helical structure near the N-terminus in trifluoroethanol and in the presence of micelles. Comparison of oligolysines with native loop residues revealed that three lysines at each terminus of a peptide corresponding to the sixth transmembrane domain of the alpha-factor receptor resulted in better aqueous solubility and greater helicity than the native loop residues.     

Structural requirements for protein N-glycosylation. Influence of acceptor peptides on cotranslational glycosylation of yeast invertase and site-directed mutagenesis around a sequon sequence

To understand better the structural requirements of the protein moiety important for N-glycosylation, we have examined the influence of proline residues with respect to their position around the consensus sequence (or sequon) Asn-Xaa-Ser/Thr. In the first part of the paper, experiments are described using a cell-free translation/glycosylation system from reticulocytes supplemented with dog pancreas microsomes to test the ability of potential acceptor peptides to interfere with glycosylation of nascent yeast invertase chains. It was found that peptides, being acceptors for oligosaccharide transferase in vitro, inhibit cotranslational glycosylation, whereas nonacceptors have no effect. Acceptor peptides do not abolish translocation of nascent chains into the endoplasmic reticulum. Results obtained with proline-containing peptides are compatible with the notion that a proline residue in an N-terminal position of a potential glycosylation site does not interfere with glycosylation, whereas in the position Xaa or at the C-terminal of the sequon, proline prevents and does not favour oligosaccharide transfer, respectively. This statement was further substantiated by in vivo studies using site-directed mutagenesis to introduce a proline residue at the C-terminal of a selected glycosylation site of invertase. Expression of this mutation in three different systems, in yeast cells, frog oocytes and by cell-free translation/glycosylation in reticulocytes supplemented with dog pancreas microsomes, leads to an inhibition of glycosylation with both qualitative and quantitative differences. This may indicate that host specific factors also contribute to glycosylation.     

Experimental conformational energy maps of proteins and peptides

We have presented an extensive analysis of the peptide backbone dihedral angles in the PDB structures and computed experimental Ramachandran plots for their distributions seen under a various constraints on X‐ray resolution, representativeness at different sequence identity percentages, and hydrogen bonding distances. These experimental distributions have been converted into isoenergy contour plots using the approach employed previously by F. M. Pohl. This has led to the identification of energetically favored minima in the Ramachandran (? , ψ ) plots in which global minima are predominantly observed either in the right‐handed α‐helical or the polyproline II regions. Further, we have identified low energy pathways for transitions between various minima in the (? ,ψ ) plots. We have compared and presented the experimental plots with published theoretical plots obtained from both molecular mechanics and quantum mechanical approaches. In addition, we have developed and employed a root mean square deviation (RMSD) metric for isoenergy contours in various ranges, as a measure (in kcal.mol^?1) to compare any two plots and determine the extent of correlation and similarity between their isoenergy contours. In general, we observe a greater degree of compatibility with experimental plots for energy maps obtained from molecular mechanics methods compared to most quantum mechanical methods. The experimental energy plots we have investigated could be helpful in refining protein structures obtained from X‐ray, NMR, and electron microscopy and in refining force field parameters to enable simulations of peptide and protein structures that have higher degree of consistency with experiments. Proteins 2017; 85:979–1001. © 2017 Wiley Periodicals, Inc.     

Studies of phosphorescent probes for proteins

1. Certain capabilities and limitations of using bound phosphorescent chromophores to study protein structure were investigated. Carbonic anhydrase inhibitors with three different arrangements of singlet and triplet energy levels relative to those of tryptophan were used to determine their ability to transfer triplet energy. 2. Ligands representing each of the three spectroscopic energy level arrangements were found to exhibit triplet-triplet energy transfer with a tryptophan residue at the active site of carbonic anhydrase. This greatly increases the number of ligands which may be useful as phosphorescent probes. 3. The efficiency of energy transfer occurs to varying degrees depending upon the inhibitor. This is a potential source of data for determining the position of the ligand in the binding site.     

Artificial binding proteins (Affitins) as probes for conformational changes in secretin PulD

The DNA-binding protein Sac7d was previously modified to bind with high affinity to the N domain of the outer membrane secretin PulD from the bacterium Klebsiella oxytoca. Here, we show that binding of the Sac7d derivatives (affitins) to PulD is sensitive to conformational changes caused by denaturant and by the zwitterionic detergent Zwittergent 3-14 routinely used to extract secretins from outer membranes. This sensitivity to the conformational state of PulD allowed us to use the affitins as probes for the native structure of PulD and to devise protocols for examining in vitro synthesized protein in nonionic detergent and for the affinity purification of native PulD using affitins as ligands. When fused to periplasmic PhoA, three affitins inhibited PulD multimerization in vivo and caused loss of function. In two cases, this was likely to be due to dimerization of the affitin by the bound PhoA, as the effect was absent when the affitins were fused to monomeric MalE. In the third case, the MalE and PhoA moieties probably interfered sterically with PulD protomer interactions and, thereby, inhibited multimerization. None of the affitins tested interacted with PulD at sites of protomer interaction or blocked the secretin channel through which exoproteins cross the outer membrane in the Type II secretion pathway of which PulD is a key component.     

Azobenzene as photoresponsive conformational switch in cyclic peptides.

Over the last decades azobenzene has been the most widely used optical trigger for the synthesis of photoresponsive systems ranging from poly-alpha-amino acids to innovative materials with light-controlled mechanical and optical properties. More recently, its use in form of appropriate derivatives allowed to generate cyclic peptide structures of constraint conformational space and thus to exploit its reversible photoisomerization to induce well defined transitions between different conformational states. These can be characterized in detail in both photostationary states making such systems ideal substrates for ultrafast spectroscopic analysis of conformational transitions. Moreover, the changes in biophysical properties that occur as a consequence of the different conformational states can be exploited for a photo-control of a large variety of molecular recognition processes.     

Fluorescent proteins as proteomic probes

Protein complexes mediate the majority of cellular processes. Knowledge of the localization and composition of such complexes provides key insights into their functions. Although green fluorescent protein (GFP) has been widely applied for in vivo visualization of proteins, it has been relatively little used as a tool for the isolation of protein complexes. Here we describe the use of the standard GFP tag to both visualize proteins in living cells and capture their interactions via a simple immunoaffinity purification procedure. We applied this method to the analysis of a variety of endogenous protein complexes from different eukaryotic cells. We show that efficient isolations can be achieved in 5-60 min. This rapid purification helps preserve protein complexes close to their original state in the cell and minimizes nonspecific interactions. Given the wide use and availability of GFP-tagged protein reagents, the present method should greatly facilitate the elucidation of many cellular processes.     

A critical evaluation of the conformational requirements of fusogenic peptides in membranes

It is generally assumed that fusogenic peptides would require a certain conformation, which triggers or participates in the rate-determining step of membrane fusion. Previous structure analyses of the viral fusion peptide from gp41 of HIV-1 have yielded contradictory results, showing either an α-helical or a β-stranded conformation under different conditions. To find out whether either of these conformations is relevant in the actual fusion process, we have placed sterically demanding substitutions into the fusion peptide FP23 to prevent or partially inhibit folding and self-assembly. A single substitution of either D- or L-CF₃-phenylglycine was introduced in different positions of the sequence, and the capability of these peptide analogues to fuse large unilamellar vesicles was monitored by lipid mixing and dynamic light scattering. If fusion proceeds via a β-stranded oligomer, then the D- and L-epimers are expected to differ systematically in their activity, since the D-epimers should be unable to form β-structures due to sterical hindrance. If an α-helical conformation is relevant for fusion, then the D-epimers would be slightly disfavoured compared to the L-forms, hence a small systematic difference in fusion activity should be observed. Interestingly, we find that (1) all D- and L-epimers are fusogenically active, though to different extents compared to the wild type, and – most importantly – (ii) there is no systematic preference for either the D- or L-forms. We therefore suggest that a well-structured α-helical peptide conformation or a β-stranded oligomeric assembly can be excluded as the rate-determining state. Instead, fusion appears to involve conformationally disordered peptides with a pronounced structural plasticity. Dedicated to Prof. K. Arnold on the occasion of this 65th birthday.     

Optimal protocol and trajectory visualization for conformational searches of peptides and proteins.

Conformational searches by molecular dynamics and different types of Monte Carlo or build-up methods usually aim to find the lowest-energy conformation. However, this is often misleading, as the energy functions used in conformational calculations are imprecise. For instance, though positions of local minima defined by the repulsive part of the Lennard-Jones potential are usually altered only slightly by functional modification, the relative depths of the minima could change significantly. Thus, the purpose of conformational searches and, correspondingly, performance criteria should be reformulated and appropriate methods found to extract different local minima from the search trajectory and allow visualization in the search space. Attempts at convergence to the lowest-energy structure should be replaced with efforts to visit a maximum number of different local energy minima with energies within a certain range. We use this quantitative criterion consistently to evaluate performances of different search procedures. To utilize information generated in the course of simulation, a "stack" of low energy conformations is created and stored. It keeps track of variables and visit numbers for the best representatives of different conformational families. To visualize the search, projection of multidimensional walks onto a principal plane defined by a set of reference structures is used. With Met-enkephalin as a structural example and a Monte Carlo procedure combined with energy minimization (MCM) as a basic search method, we analyzed the influence on search efficiency of different characteristics as temperature schedules, the step size for variable modification, constrained random step and response mechanisms to search difficulties. Simulated annealing MCM had comparable efficiency with MCM at constant and elevated temperature (about 600 K). Constraining the randomized choice of side-chain chi angles to optimal values (rotamers) on every MCM step did not improve, but rather worsened, the search efficiency. Two low-energy Met-enkephalin conformations with parallel Tyr1 and Phe4 rings, a gamma-turn around the Gly2 residue, and Phe4 and Met5 side-chains forming together a compact hydrophobic cluster were found and are suggested as possible structural candidates for interaction with a receptor or a membrane.     

Substrate specificity of protein kinase C. Use of synthetic peptides corresponding to physiological sites as probes for substrate recognition requirements

Although the Ca2+/phospholipid-dependent protein kinase, protein kinase C, has a broad substrate specificity in vitro, the enzyme appears considerably less promiscuous in vivo. To date only a handful of proteins have been identified as physiological substrates for this protein kinase. In order to determine the basis for this selectivity for substrates in intact cells, we have probed the substrate primary sequence requirements of protein kinase C using synthetic peptides corresponding to sites of phosphorylation from four of the known physiological substrates. We have also identified the acetylated N-terminal serine of chick muscle lactate dehydrogenase as an in vitro site of phosphorylation for this protein kinase. These comparative studies have demonstrated that, in vivo, the enzyme exhibits a preference for one basic residue C-terminal to the phosphorylatable residue, as in the sequence: Ser/Thr-Xaa-Lys/Arg, where Xaa is usually an uncharged residue. Additional basic residues, both N and C-terminal to the target amino acid, enhance the Vmax and Km parameters of phosphorylation. None of the peptides based on physiological phosphorylation sites of protein kinase C was an efficient substrate of cAMP-dependent protein kinase, emphasizing the distinct site-recognition selectivities of these two pleiotropic protein kinases. The favorable kinetic parameters of several of the synthetic peptides, coupled with their selectivity for phosphorylation by protein kinase C, will facilitate the assay of this enzyme in the presence of other protein kinases in tissue and cell extracts.     

Synthetic peptides as probes of plant cell signalling

Synthetic peptides have found increasing use in dissecting cell signalling pathways and have been employed as synthetic antigens, protein kinase and protease substrates. Recently, it has become evident that relatively short (10–30mer) peptides are able to mimic that part of the signalling protein to which their sequence corresponds. In particular, peptides corresponding to the C-terminus of Zea mays auxin binding protein, ZmABP1, were able to modulate ion channel function within Vicia guard cells. In this report, GTPS binding to NaCl-washed Zea microsomal membranes is shown to be stimulated by peptide A6.2, corresponding to the C-terminal 16 residues of ZmABP1, only when the membranes are reconstituted with soluble Zea protein fractions containing GP1 and G₀ homologues.     

Narrowing the conformational space sampled by two-domain proteins with paramagnetic probes in both domains

Calmodulin is a two-domain protein which in solution can adopt a variety of conformations upon reorientation of its domains. The maximum occurrence (MO) of a set of calmodulin conformations that are representative of the overall conformational space possibly sampled by the protein, has been calculated from the paramagnetism-based restraints. These restraints were measured after inclusion of a lanthanide binding tag in the C-terminal domain to supplement the data obtained by substitution of three paramagnetic lanthanide ions to the calcium ion in the second calcium binding loop of the N-terminal domain. The analysis shows that the availability of paramagnetic restraints arising from metal ions placed on both domains, reduces the MO of the conformations to different extents, thereby helping to identify those conformations that can be mostly sampled by the protein.