An improved protocol for coupling synthetic peptides to carrier proteins for antibody production using DMF to solubilize peptides.

We present an improved protocol for coupling synthetic peptides to carrier proteins. In this protocol, dimethyl-formamide is used as the solvent to solubilize peptides instead of phosphate-buffered saline (PBS) or 6 M guanidine-HCl/0.01 M phosphate buffer (pH 7). Additionally, the last desalting or dialyzing step to remove uncoupled peptides as in the traditional method is eliminated. Finally, 3 ml of 0.1 M ammonium bicarbonate is added to the carrier protein conjugated peptide solution to help the lyophilization process. Coupling of Cys-containing synthetic peptides to keyhole limpet hemocyanin or bovine serum albumin using m-maleimidobenzoyl-N-hydroxysuccinimide ester are used as the test cases. This method produces high-quality antipeptide antibodies. Also, compared to the traditional method, this procedure is simpler and useful for peptides with solubility problems in PBS or 6 M guanidine-HCl.     

Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes

Conformational aspects of N-glycosylation have been investigated with a series of proline-containing peptides as molecular probes. The results demonstrate that, depending on the position of the imino acid in the peptide chain, dramatic alterations of glycosylation rates are produced, pointing to a critical contribution of the amino acids framing the 'marker sequence' triplet Asn-Xaa-Thr(Ser) on the formation of a potential sugar-attachment site. No glycosyl transfer at all was detectable to those peptides containing a proline residue either in position Xaa or in the next position beyond the threonine of the Asn-sequon on the C-terminal side, whereas the hexapeptide Pro-Asn-Gly-Thr-Ala-Val was glycosylated at a high rate. (Emboldened residues denote the 'marker sequence' that is identical in all the peptides; italicized residues distinguish the positions of proline in the various peptides.) Studies with space-filling models reveal that the lack of glycosyl-acceptor capabilities of Ala(Pro)-Asn-Gly-Thr-Pro-Val might be directly related to their inability to adopt and/or stabilize a turn or loop conformation which permits the catalytically essential interaction between the hydroxy amino acid and the asparagine residue within the 'marker sequence' [Bause & Legler (1981) Biochem. J. 195, 639-644]. This conclusion is supported by circular-dichroism spectroscopic data, which suggest structure-forming potentials in this type of non-acceptor peptides dominating over those that favour the induction of an appropriate sugar-attachment site in the acceptor peptides. The lack of acceptor properties of Tyr-Asn-Pro-Thr-Ser-Val indicates that even small modifications in the 'recognition' pattern are not tolerated by the N-glycosyltransferases.     

Synthesis and conformational studies of peptides fromE. coli pilus proteins recognized by the chaperone PapD

Summary Adhesive pili in uropathogenicE. coli are composed of a few different types of proteins which are assembled into the pilus by the chaperone PapD. Peptides from the C-terminus of these pilus proteins have been prepared by Fmoc solid-phase synthesis. Use of a polyethyleneglycol polystyrene resin was found to be essential for successful synthesis. The conformational propensities of the peptides were analyzed by CD and¹H NMR spectroscopy, with special focus on PapG296-314 from the pilus adhesin which has previously showed the tightest binding to PapD. PapG296-314 was found to be flexible in different solutions, but with a significant propensity to adopt a -conformation. Interestingly the peptide is bound as a -strand in a crystalline complex with PapD. The peptides from three other pilus proteins could only be investigated in trifluoroethanol wher they displayed considerable -helicity in contrast to PapG296-314. These results suggest that conformational factors provide part of the explanation for the differential binding of pilus-related peptides to PapD.Abbreviations CD circular dichroism - CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate - COSY two-dimensional correlated spectroscopy - DMF dimethylformamide - DMSO dimethylsulfoxide - FAB-MS fast-atom-bombardment mass spectroscopy - Fmoc 9-fluorenylmethoxycarbonyl - Gal galactose - HOBt 1-hydroxybenzotriazole - MeCN acetonitrile - MSNT 1-(2-mesitylenesulfonyl)-3-nitro-1,2,4-triazole - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser enhancement spectroscopy - PEG-PS resin polyethyleneglycol polystyrene resin - ROESY two-dimensional rotating frame nuclear Overhauser enhancement spectroscopy - SDS sodium dodecylsulfate - TFA trifluoroacetic acid - TFE 2,2,2-trifluoroethanol - TOCSY two-dimensional total correlation spectroscopy These authors have made equal contributions to the work presented.correspondence should be addressed.     

Leap-dynamics: efficient sampling of conformational space of proteins and peptides in solution

Glucose-6-phosphate dehydrogenase (G6PD) plays an important role in cellular redox homeostasis, which is crucial for cell survival. In the present study, we found that G6PD status determines the response of cells exposed to nitric oxide (NO) donor. Treatment with NO donor, sodium nitroprusside (SNP), caused apoptosis in G6PD-deficient human foreskin fibroblasts (HFF1), whereas it was growth stimulatory in the normal counterpart (HFF3). Such effects were abolished by NO scavengers like hemoglobin. Ectopic expression of G6PD in HFF1 cells switched the cellular response to NO from apoptosis to growth stimulation. Experiments with 1H-?1,2,4?xadiazolo?4, 3-aquinoxalin-1-one and 8-bromo-cGMP showed that the effects of NO on HFF1 and HFF3 cells were independent of cGMP signalling pathway. Intriguingly, trolox prevented the SNP-induced apoptosis in HFF1 cells. These data demonstrate that G6PD plays a critical role in regulation of cell growth and survival.     

Leap-dynamics: efficient sampling of conformational space of proteins and peptides in solution

A molecular simulation scheme, called Leap-dynamics, that provides efficient sampling of protein conformational space in solution is presented. The scheme is a combined approach using a fast sampling method, imposing conformational 'leaps' to force the system over energy barriers, and molecular dynamics (MD) for refinement. The presence of solvent is approximated by a potential of mean force depending on the solvent accessible surface area. The method has been successfully applied to N-acetyl-L-alanine-N-methylamide (alanine dipeptide), sampling experimentally observed conformations inaccessible to MD alone under the chosen conditions. The method predicts correctly the increased partial flexibility of the mutant Y35G compared to native bovine pancreatic trypsin inhibitor. In particular, the improvement over MD consists of the detection of conformational flexibility that corresponds closely to slow motions identified by nuclear magnetic resonance techniques.     

Amino acid conformational preferences and solvation of polar backbone atoms in peptides and proteins

Amino acids in peptides and proteins display distinct preferences for alpha-helical, beta-strand, and other conformational states. Various physicochemical reasons for these preferences have been suggested: conformational entropy, steric factors, hydrophobic effect, and backbone electrostatics; however, the issue remains controversial. It has been proposed recently that the side-chain-dependent solvent screening of the local and non-local backbone electrostatic interactions primarily determines the preferences not only for the alpha-helical but also for all other main-chain conformational states. Side-chains modulate the electrostatic screening of backbone interactions by excluding the solvent from the vicinity of main-chain polar atoms. The deficiency of this electrostatic screening model of amino acid preferences is that the relationships between the main-chain electrostatics and the amino acid preferences have been demonstrated for a limited set of six non-polar amino acid types in proteins only. Here, these relationships are determined for all amino acid types in tripeptides, dekapeptides, and proteins. The solvation free energies of polar backbone atoms are approximated by the electrostatic contributions calculated by the finite difference Poisson-Boltzmann and the Langevin dipoles methods. The results show that the average solvation free energy of main-chain polar atoms depends strongly on backbone conformation, shape of side-chains, and exposure to solvent. The equilibrium between the low-energy beta-strand conformation of an amino acid (anti-parallel alignment of backbone dipole moments) and the high-energy alpha conformation (parallel alignment of backbone dipole moments) is strongly influenced by the solvation of backbone polar atoms. The free energy cost of reaching the alpha conformation is by approximately 1.5 kcal/mol smaller for residues with short side-chains than it is for the large beta-branched amino acid residues. This free energy difference is comparable to those obtained experimentally by mutation studies and is thus large enough to account for the distinct preferences of amino acid residues. The screening coefficients gamma(local)(r) and gamma(non-local)(r) correlate with the solvation effects for 19 amino acid types with the coefficients between 0.698 to 0.851, depending on the type of calculation and on the set of point atomic charges used. The screening coefficients gamma(local)(r) increase with the level of burial of amino acids in proteins, converging to 1.0 for the completely buried amino acid residues. The backbone solvation free energies of amino acid residues involved in strong hydrogen bonding (for example: in the middle of an alpha-helix) are small. The hydrogen bonded backbone is thus more hydrophobic than the peptide groups in random coil. The alpha-helix forming preference of alanine is attributed to the relatively small free energy cost of reaching the high-energy alpha-helix conformation. These results confirm that the side-chain-dependent solvent screening of the backbone electrostatic interactions is the dominant factor in determining amino acid conformational preferences.     

Optimal conditions for the use of protein L-isoaspartyl methyltransferase in assessing the isoaspartate content of peptides and proteins.

Protein L-isoaspartyl methyltransferase provides a basis for enzymatic measurement of atypical, isoaspartyl linkages which make a major contribution to protein microheterogeneity. The low Vmax of the methyltransferase reaction and the instability of the methyl ester can hinder accurate determinations, and different laboratories using different conditions have achieved discrepant values for the isoaspartate content of the same proteins. To investigate the effects of these conditions, and to optimize the assay, isoaspartyl delta sleep-inducing peptide was methylated under a variety of conditions. We found that 1 microM methyltransferase was required to obtain stoichiometric modification of 2 microM peptide in 40-min reactions at pH 6.2 and 30 degrees C. A computer model utilizing kinetic constants obtained from studies on initial rates of methylation predicted the same requirement for enzyme concentration. Carrier protein was necessary for optimal methyltransferase activity at enzyme concentrations below 0.4 microM. Stoichiometric methylation required concentrations of S-adenosylmethionine to be in substantial excess over those of peptide; 50 microM S-adenosylmethionine is the minimum needed for complete modification of 10 microM peptide. Spontaneous demethylation was significant under all conditions tested, so that the methyl ester itself never reached a ratio of 1 mol/mol of total peptide. These results demonstrate that the most accurate measurements of isoaspartate are obtained when reactions are carried out at low peptide concentrations, high S-adenosylmethionine concentrations, and high enzyme concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prediction of homologous protein structures based on conformational searches and energetics

A "knowledge-based" method of predicting the unknown structure of a protein from a homologous known structure using energetics to determine a sidechain conformation is proposed. The method consists of exchanging the residues in the known structure for the sequence of the unknown protein. Then a conformational search with molecular mechanics energy minimization is done on the exchanged residues. The lowest energy conformer is the one picked to be the predicted structure. In the structure of bovine trypsin, the importance of including a solvation energy term in the search is demonstrated for solvent accessible residues, while molecular mechanics alone is enough to correctly predict the conformation of internal residues. The correctness of the model is assessed by a volume error overlap of the predicted structure compared to the crystal structure. Finally, the structure of rat trypsin is predicted from the crystal structure of bovine trypsin. The sequences of these two proteins are 74% identical and all of the significant changes between them are on external residues. Thus, the inclusion of solvation energy in the conformational search is necessary to accurately predict the structure of the exchanged residues.     

GPMAW--a software tool for analyzing proteins and peptides.

General Protein/Mass Analysis for Windows (GPMAW) is a valuable piece of software for any molecular biologist, biochemist or mass spectrometrist wishing to analyze protein or peptide sequences. All steps from the acquisition of protein sequence from a built-in web interface, to proteolytic digests, theoretical peptide fragmentation, detailed annotation of sequences and secondary structure prediction, can be performed rapidly and intuitively without first having to spend days reading manuals.     

Theoretical conformational analysis of brain peptides. beta-Melanocyte-stimulating hormone

Using a semi-empirical method, an a priori conformational analysis of the octadecapeptide beta-melanocyte-stimulating hormone (beta-MSH) was carried out. The spatial structure of beta-MSH can be described by eight low-energy conformations, yielded by combinations of the most stable states of the respective free fragments. Calculations produced the values of all dihedral angles of the backbones and side chains of these forms as well as intra- and inter-residue interaction energies.     

ATP-induced conformational transition of denatured proteins.

Although the conformational change occurring in proteins upon ATP binding is important in many biological reactions, the mechanism by which ATP binding induces the conformational change is unknown. We found that ATP induces acid-unfolded (pH 2) ferricytochrome c or apomyoglobin to adopt a compact structure with a significant amount of alpha-helix and increased hydrophobicity. A very similar conformational transition was observed at neutral pH for an amphiphilic model polypeptide. The effectiveness of various adenine nucleotides in inducing the conformational transition was found to be proportional to their phosphate group contents, i.e., adenosine tetraphosphate greater than ATP greater than ADP greater than AMP. These results should be important when considering the mechanism of the ATP-induced conformational change in proteins during various biological reactions.     

Algorithm for predicting functionally equivalent proteins from BLAST and HMMER searches

In order to predict biologically significant attributes such as function from protein sequences, searching against large databases for homologous proteins is a common practice. In particular, BLAST and HMMER are widely used in a variety of biological fields. However, sequencehomologous proteins determined by BLAST and proteins having the same domains predicted by HMMER are not always functionally equivalent, even though their sequences are aligning with high similarity. Thus, accurate assignment of functionally equivalent proteins from aligned sequences remains a challenge in bioinformatics. We have developed the FEP-BH algorithm to predict functionally equivalent proteins from protein-protein pairs identified by BLAST and from protein-domain pairs predicted by HMMER. When examined against domain classes of the Pfam-A seed database, FEP-BH showed 71.53% accuracy, whereas BLAST and HMMER were 57.72% and 36.62%, respectively. We expect that the FEP-BH algorithm will be effective in predicting functionally equivalent proteins from BLAST and HMMER outputs and will also suit biologists who want to search out functionally equivalent proteins from among sequence-homologous proteins.     

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.     

The nasal delivery of peptides and proteins.

Many drugs of the future will be therapeutically active peptides and proteins developed through recombinant-DNA technology. A major factor limiting their exploitation is the current lack of appropriate non-parenteral delivery systems. Nasal systems incorporating absorption enhancers may provide a convenient, efficient means of administering protein and peptide therapeutics.     

Reduction of sulfoxides in peptides and proteins.

The reduction of methionine sulfoxide to methionine in peptides and proteins has been systematically investigated in terms of specific reducing agent, concentration of reducing agent, temperature, pH of the solution, and the presence of denaturing agents. While several of the reagents examined had a greater rate of reduction, N-methylmercaptoacetamide was found to be the reducing agent of choice as it was the reagent with the highest rate of reduction having no adverse interaction with other residues in peptides and proteins. Its rate of reduction increased until its concentration reached approximately 50% ($\text{v}\text{v}$). Its reducing ability was relatively independent of pH changes but decreased with increases in acetic acid concentration. Using this reagent under acid, neutral, or basic conditions at a concentration of 0.7–2.8 m, methionine sulfoxide can be completely reduced to methionine in peptides and proteins at 37°C in 12 to 24 h. The sulfoxide form of S-carbamoylmethylcysteine in peptide and proteins takes approximately five times longer to reduce than methionine sulfoxide.     

Exhaustive conformational search and simulated annealing for models of lattice peptides

We consider simple lattice models for short peptide chains whose states can be exhaustively enumerated to find the lowest energy conformation. Using these exact results and numerical simulations, we compute the distributions for the mean time t_N, required to find the global minimum energy state by simulated annealing (SA), as a function of N, the number of units in the chain. On the basis of scaling arguments, the time t_N, to find the global minimum energy of longer chains, beyond the range covered by exhaustive enumeration, can be estimated. On the basis of the observed exponential increase in folding time of the standard SA algorithms, it is imperative that better algorithms be found for minimizing longer chains. © 1993 John Wiley & Sons, Inc.