Binding Of S-peptide Research Articles

Comparative Molecular Dynamics Analysis of RNase-S Complex Formation

Limited proteolysis of RNase-A yields a short N-terminal S-peptide segment and the larger S-protein. Binding of S-peptide to S-protein results in the formation of an enzymatically active RNase-S protein. S-peptide undergoes a transition from intrinsic disorder to an ordered helical state upon association with S-protein to form RNase-S and is an excellent model system to study coupled folding and binding. To better understand the dynamics of the RNases-S complex and its isolated partners, comparative molecular dynamics simulations have been performed. In agreement with experiment, we find significant conformational fluctuations of the isolated S-peptide compatible with a disordered regime and only little residual helical structure. In the RNase-S complex, the N-terminal helix of S-peptide unfolds and refolds repeatedly on the microsecond timescale, indicating that the α-helical structure is only part of the equilibrium regime for these residues whereas the C-terminal residues are confined to the helical conformation that is found in the x-ray structure. This is also in line with systematic, in silico Alanine scanning free-energy simulations, which indicate that the major contribution to complex stability emerges from the C-terminal helical turn, consisting of residues 8–13 in S-peptide whereas the N-terminal S-peptide residues 1–7 make only minor contributions. Comparative simulations of S-protein in the presence and absence of S-peptide reveal that the isolated S-protein is significantly more flexible than in the complex, and undergoes a global pincerlike conformational change that narrows the S-peptide binding cleft. The narrowed binding cleft adds a barrier for complex formation likely influencing the binding kinetics. This conformational change is reversed by S-peptide association, which also stabilizes conformational fluctuations in S-protein. Such global motions associated with binding are also likely to play a role for other coupled peptide folding and binding processes at peptide binding regions on protein surfaces.

Temperature dependence of ligand–protein complex formation as reflected by saturation transfer difference NMR experiments

We show that temperature is an important parameter for the sensitivity of saturation transfer difference (STD) spectroscopy. A decreased intensity of STD signals is observed for lactose binding to growth-regulatory galectin7 (p53-induced gene 1), as well as for nucleotide binding to annexin A6, when the temperature is increased from 281 to 298-310 K. Opposite temperature effects on STD intensity are observed for S-peptide binding to S-protein to reconstitute RNase S. However, the STD signals for tryptophan binding to downstream regulatory element antagonist modulator of the human prodynorphin gene (DREAM)are relatively unaffected between 281 and 298 K. The known kinetics of the binding of ATP by the uncoupling protein from brown adipose tissue mitochondria (UCP1) predicted an observable STD at 310 K, but rapid sample degradation limits the experiments to much lower temperatures. Temperature strongly influences the kinetics and affinity constant of various types of complex formation and in so doing influences the observed STD effects. Therefore, temperature can be exploited to facilitate the optimization of STD-based applications, and at the same time minimize the number of test samples. STD-based screening protocols to detect new target-specific compounds may yield a larger number of potential ligands if screened at various temperatures.

Study of the ribonuclease-S-protein-peptide complex using a radical probe and electrospray ionization mass spectrometry.

The interaction between ribonuclease (RNase) S-protein and S-peptide is examined by studying their limited oxidation within the RNase-S complex and free forms using radicals. The limited oxidation of the RNase-S complex and each component is effected through their reaction with a high flux of oxygen-based radicals generated by an electrical discharge within an electrospray ion source. Their exposure to radicals occurs on short millisecond time scales and has been consistently found not to cause any measurable structural damage or conformational change to proteins in a number of published reports. Consistent with these studies, S-peptide is preferentially protected from reactions with radicals under conditions in which it is bound to S-protein. Conversely, a region of S-protein comprising residues 96-100 constitutes the S-peptide binding domain based on its diminished reactivity with radicals within the RNase-S complex over the free S-protein. The results, for the first time, demonstrate the use of radicals generated by an electrical discharge to study protein complexes.

Kinetic characterization of ribonuclease S mutants containing photoisomerizable phenylazophenylalanine residues.

Incorporation of the photoisomerizable amino acid phenylazophenylalanine (PAP) into enzyme structures has been proposed as a strategy for photoswitching enzyme activity. To evaluate the strengths and limitations of this approach to enzyme photo-control, we performed a kinetic analysis of RNase S analogues containing PAP in positions 4, 7, 8, 10, 11 or 13. For an enzyme containing a single PAP group, the maximum extent of photoconversion (between approximately 96% trans/4% cis and 10% trans/90% cis under standard conditions) sets a limit on the maximum fold change in the initial rate of approximately 25-fold, if the cis form is the more active isomer, and approximately 10-fold if the trans form is more active. This extent of photoswitching was not realized in the present case because the effects of photoisomerization on kinetic constants were small and distributed among effects on S-peptide binding, substrate binding and the rate of the chemical step. These results suggest that photoisomerization could substantially alter enzyme kinetic constants but that a directed combinatorial approach might be required for realizing maximal photo-control in such systems. The limit set by the extent of photoconversion might be overcome by coupling multiple PAP groups to one enzyme or by altering the behaviour of a system that required oligomerization for activity.

High affinity RNase S-peptide variants obtained by phage display have a novel "hot-spot" of binding energy.

Using phage display mutagenesis, high affinity variants of RNase S-peptide were produced that bind to RNase S-protein over 100-fold more tightly than the wild type S-peptide. The S-peptide: S-protein interface was further characterized using "biased" phage display libraries, where each targeted residue was constrained to be either polar or nonpolar. The use of these tailored libraries placed constraints on the type of interactions present during affinity maturation process and allowed more amino acids to be randomized simultaneously. These results, in conjunction with kinetic association and dissociation constants determined by surface plasmon resonance (SPR), highlight the role of a single mutation (A5W) in increasing S-peptide binding affinity. High affinity S-peptide variants were only identified when tryptophan was present in the phage display library at position 5, suggesting that this residue is a "hot-spot" of binding energy in the high affinity variants. Analysis of SPR data in the presence of denaturant suggests that the increased affinity is a result of increased hydrophobic interactions in the transition state rather than a stabilization of helical structure.

Effects of binding of S-peptide and 2′-cytidine monophosphate on hydrogen exchange from the S-protein component of ribonuclease S: The amide protons of serine 123 and valine 124

The hydrogen exchange rates of amide protons from many chain segments in S-protein, previously shown to be dramatically affected on association with S-peptide, are essentially unchanged, over a period of 150 hours at least, on binding the inhibitor 2′CMP to RNAase S. An exception was the C-terminal tetrapeptide (121–124), which is close to, but not in actual contact with, substrates or inhibitors bound at the active site of the enzyme. Due to the effect of the intrinsic exchange rates within the fragment during analysis only two protons are observed, those of serine 123 and valine 124. The intrinsic rates of these two sites are also sufficiently different that their rates of exchange in the intact protein can be separately monitored. Both rates decrease on binding S-peptide and decrease by an additional factor of ten on binding 2′CMP. The amide of Vall24 is hydrogen bonded as part of a β-sheet region in the native structure. The amide of Ser123 is not hydrogen-bonded, is directed towards the solvent, but is not accessible to it in the time average structure of the intact enzyme. The main-chain motion required to permit exchange is such that both amides are affected in the same way in spite of their different environments.

Hydrogen exchange from identified regions of the S-protein component of ribonuclease as a function of temperature, pH, and the binding of S-peptide

A medium resolution hydrogen exchange method (Rosa & Richards, 1979) has been used to measure the average rates of amide hydrogen exchange for known segments of the S-protein portion of ribonuclease-S. The analytical procedure permitted exchange rates to be monitored for seven S-protein fragments distributed throughout the structure, including regions of α-helix and β-sheet. Kinetics were measured as a function of pH, temperature and S-peptide binding. The pH dependence of exchange from isolated S-protein between pH 2·8 and pH 7·0 was found to deviate significantly from a first-order dependence on hydroxide ion concentration. The protection against exchange with increasing pH appeared to be closely related to the electrostatic stabilization of S-protein. It is suggested that such favorable electrostatic interactions result in increased energy barriers to the conformational fluctuations that provide solvent access to the time-average crystallographic structure. This explanation of the observed correlation between stability and exchange kinetics is also consistent with the calculated apparent activation energies for exchange from S-protein between 5·5 and 20 °C. S-peptide binding dramatically slows exchange from many S-protein sites, even those distant from the area of S-peptide contact. Interestingly, the effects of complex formation are not evenly propagated throughout S-protein. The most significantly perturbed sites (≥10 3-fold reduction in exchange rate constants) lie within fragments derived from regions of secondary structure. Exchange from several other fragments is not significantly affected. The S-peptide—S-protein dissociation constant at neutral pH is so small that the measured exchange must have occurred from the complex and not from the dissociated parts.

The preparation and primary structure of S-peptides from different pancreatic ribonucleases

In 1955, Richards [l] described the isolation of ‘an active intermediate produced during the digestion of ribonuclease by subtilisin’. The characterisation and separation of the non-covalently linked components was described 4 years later [2] . Ribonuclease S* possesses full enzymatic activity and the same holds for the enzyme reconstituted from S-peptide and S-protein. The involvement of S-peptide residues in the binding of S-peptide to S-protein and in the enzymatic activity of the reconstituted RNase S’ has been studied by using synthetic S-peptide analogs [3,4] the cleavage by subtilisin takes place in an external loop. Klee [5] and Gold [6] did not succeed in cleaving the RNases from rat and snapping turtle with subtilisin. In this study, we present the successful cleavage with subtilisin Carlsberg of the RNase from goat, brindled gnu, giraffe, reindeer, dromedary, and red kangaroo and the isolation of the corresponding S-peptides. Differences in the observed behaviour are compared with predicted differences in conformation.

Thermodynamics of the binding of S-peptide to S-protein to form ribonuclease S..

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThermodynamics of the binding of S-peptide to S-protein to form ribonuclease S'Julian M. Sturtevant, Ruby P. Hearn, Frederic M. Richards, and Gerald D. WattCite this: Biochemistry 1971, 10, 5, 806–817Publication Date (Print):March 1, 1971Publication History Published online1 May 2002Published inissue 1 March 1971https://doi.org/10.1021/bi00781a013RIGHTS & PERMISSIONSArticle Views141Altmetric-Citations82LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

A convenient method for the preparation of S-peptide from bovine pancreatic ribonuclease

A medium resolution hydrogen exchange method (Rosa & Richards, 1979) has been used to measure the average rates of amide hydrogen exchange for known segments of the S-protein portion of ribonuclease-S. The analytical procedure permitted exchange rates to be monitored for seven S-protein fragments distributed throughout the structure, including regions of α-helix and β-sheet. Kinetics were measured as a function of pH, temperature and S-peptide binding.The pH dependence of exchange from isolated S-protein between pH 2·8 and pH 7·0 was found to deviate significantly from a first-order dependence on hydroxide ion concentration. The protection against exchange with increasing pH appeared to be closely related to the electrostatic stabilization of S-protein. It is suggested that such favorable electrostatic interactions result in increased energy barriers to the conformational fluctuations that provide solvent access to the time-average crystallographic structure. This explanation of the observed correlation between stability and exchange kinetics is also consistent with the calculated apparent activation energies for exchange from S-protein between 5·5 and 20 °C.S-peptide binding dramatically slows exchange from many S-protein sites, even those distant from the area of S-peptide contact. Interestingly, the effects of complex formation are not evenly propagated throughout S-protein. The most significantly perturbed sites (≥103-fold reduction in exchange rate constants) lie within fragments derived from regions of secondary structure. Exchange from several other fragments is not significantly affected. The S-peptide—S-protein dissociation constant at neutral pH is so small that the measured exchange must have occurred from the complex and not from the dissociated parts.