The development of biomolecular Raman optical activity spectroscopy

Following its first observation over 40 years ago, Raman optical activity (ROA), which may be measured as a small difference in the intensity of vibrational Raman scattering from chiral molecules in right- and left-circularly polarized incident light or, equivalently, the intensity of a small circularly polarized component in the scattered light using incident light of fixed polarization, has evolved into a powerful chiroptical spectroscopy for studying a large range of biomolecules in aqueous solution. The long and tortuous path leading to the first observations of ROA in biomolecules in 1989, in which the author was closely involved from the very beginning, is documented, followed by a survey of subsequent developments and applications up to the present day. Among other things, ROA provides information about motif and fold, as well as secondary structure, of proteins; solution structure of carbohydrates; polypeptide and carbohydrate structure of intact glycoproteins; new insight into structural elements present in unfolded protein sequences; and protein and nucleic acid structure of intact viruses. Quantum chemical simulations of observed Raman optical activity spectra provide the complete three-dimensional structure, together with information about conformational dynamics, of smaller biomolecules. Biomolecular ROA measurements are now routine thanks to a commercial instrument based on a novel design becoming available in 2004

Photoswitchable peptidomimetics with diarylethene building blocks

Incorporation of molecular photoswitches into peptides or proteins should allow reversible control of their structure and functions by means of photoisomerization. The aim of this study was to explore the practical possibilities of polypeptide modification using photoswitchable diarylethene-based building blocks, and to investigate the effects of the photoinduced isomerization on the structure and function of several biologically active peptides

Dynamics in unfolded polypeptide chains as model for elementary steps in protein folding

This thesis deals with the dynamics of unfolded polypeptide chains as model for the earliest steps in protein folding. Starting from an ensemble of unfolded conformations a folding polypeptide chain has to form specific backbone and side-chain interactions to reach the native state. The rate at which two defined contacts are formed on a polypeptide chain is limited by intrachain diffusion. The characterization of rate constants of intrachain contact formation in polypeptides and their dependence on length, sequence and solvent effects give new insights for an understanding of the dynamics of the earliest steps in protein folding. Until recently, little was known about absolute time scales of intramolecular contact formation in polypeptide chains. Direct measurements of fast intramolecular diffusion processes became possible with the development of fast diffusion-controlled electron transfer processes. In the presented work triplet-triplet energy transfer was used to characterize intrachain contact formation in homo-polypeptides and peptide fragments derived from natural protein sequences. The transfer of triplet electrons between the triplet donor xanthone to the triplet acceptor naphthalene is diffusion-controlled as tested by measuring its temperature and viscosity dependencies. The results suggest that triplet-triplet energy transfer from xanthone to naphthalene provides the requirement to determine absolute intramolecular contact formation rate constants in polypeptide chains. Intrachain contact formation in unstructured polypeptides is well described as a single exponential process. The loop-size dependence of the rate constants of intrachain contact formation revealed that intrachain motions over short and long distances are limited by different rate-limiting steps. In short peptide chains end-to-end contact formation is with a minimum time constant of 5-10 ns virtually independent of chain length and limited by an activation barrier of 12-16 kJ/mol. In long flexible poly(glycine-serine) peptide chains with more than twenty peptide bonds N the rate constants decrease with N-1.7±0.1 and end-to-end contact formation becomes nearly completely entropy-driven. Glycine and proline residues significantly change local intrachain dynamics compared to all other amino acids. Glycine accelerates contact formation whereas short proline containing peptides reveal complex kinetics of contact formation. Local chain dynamics are accelerated by a cis and slowed down by a trans peptidyl-prolyl bond. The effects vanish in peptide chains if the sequence contains more than five amino acids on each side of a single glycyine or a single proline residue. The dynamics of loop formation are sensitive to the nature of the solvent. Good solvents, such as denaturants slow down intrachain dynamics compared to water. The effect of solvent composition on chain dynamics indicates that the chain properties of polypeptides strongly depend on the surrounding conditions. Natural protein sequences are more complex than homo-polypeptide chains because they consist of 20 different amino acids. We determined the dynamics of loop formation in sequences derived from two proteins, carp muscle β-parvalbumin and protein G B1 domain. Compared to homo-polypeptides the intrachain dynamics in natural loop sequences are slowed down and higher activation barriers are determined. The results suggest that the dynamics of the earliest steps in protein folding are limited by significant activation barriers. The results allow us to estimate an upper time scale for rates of contact formation in unstructured peptide chains. In glycine-rich sequences, which are often found in β- hairpins and turns a first contact over 3-4 peptide bonds will be formed within 10-15 ns. For glycine-free sequences local contact formation is slowed down to 15-50 ns depending on the sequence. Due to the strong distance dependence of the rate constant of the end-to-end contact formation long-range interactions on an unfolded polypeptide chain over 50-60 peptide bonds will not be formed faster than in 500 ns

Designing Peptidomimetics

The concept of a peptidomimetic was coined about forty years ago. Since then, an enormous effort and interest has been devoted to mimic the properties of peptides with small molecules or pseudopeptides. The present report aims to review different approaches described in the past to succeed in this goal. Basically, there are two different approaches to design peptidomimetics: a medicinal chemistry approach, where parts of the peptide are successively replaced by non-peptide moieties until getting a non-peptide molecule and a biophysical approach, where a hypothesis of the bioactive form of the peptide is sketched and peptidomimetics are designed based on hanging the appropriate chemical moieties on diverse scaffolds. Although both approaches have been used in the past, the former has been more widely used to design peptidomimetics of secretory peptides, whereas the latter is nowadays getting momentum with the recent interest in designing protein-protein interaction inhibitors. The present report summarizes the relevance of the information gathered from structure-activity studies, together with a short review on the strategies used to design new peptide analogs and surrogates. In a following section there is a short discussion on the characterization of the bioactive conformation of a peptide, to continue describing the process of designing conformationally constrained analogs producing first and second generation peptidomimetics. Finally, there is a section devoted to review the use of organic scaffolds to design peptidomimetics based on the information available on the bioactive conformation of the peptide.Postprint (author's final draft

Rational Design of Protein Stability: Effect of (2S,4R)-4-Fluoroproline on the Stability and Folding Pathway of Ubiquitin

BACKGROUND: Many strategies have been employed to increase the conformational stability of proteins. The use of 4-substituted proline analogs capable to induce pre-organization in target proteins is an attractive tool to deliver an additional conformational stability without perturbing the overall protein structure. Both, peptides and proteins containing 4-fluorinated proline derivatives can be stabilized by forcing the pyrrolidine ring in its favored puckering conformation. The fluorinated pyrrolidine rings of proline can preferably stabilize either a C(γ)-exo or a C(γ)-endo ring pucker in dependence of proline chirality (4R/4S) in a complex protein structure. To examine whether this rational strategy can be generally used for protein stabilization, we have chosen human ubiquitin as a model protein which contains three proline residues displaying C(γ)-exo puckering. METHODOLOGY/PRINCIPAL FINDINGS: While (2S,4R)-4-fluoroproline ((4R)-FPro) containing ubiquitinin can be expressed in related auxotrophic Escherichia coli strain, all attempts to incorporate (2S,4S)-4-fluoroproline ((4S)-FPro) failed. Our results indicate that (4R)-FPro is favoring the C(γ)-exo conformation present in the wild type structure and stabilizes the protein structure due to a pre-organization effect. This was confirmed by thermal and guanidinium chloride-induced denaturation profile analyses, where we observed an increase in stability of -4.71 kJ·mol(-1) in the case of (4R)-FPro containing ubiquitin ((4R)-FPro-ub) compared to wild type ubiquitin (wt-ub). Expectedly, activity assays revealed that (4R)-FPro-ub retained the full biological activity compared to wt-ub. CONCLUSIONS/SIGNIFICANCE: The results fully confirm the general applicability of incorporating fluoroproline derivatives for improving protein stability. In general, a rational design strategy that enforces the natural occurring proline puckering conformation can be used to stabilize the desired target protein

Total synthesis of a virotoxin and analogs for conformational studies

This dissertation describes the first total synthesis of alloviroidin in trace amounts, along with that of three analogs containing L-proline (Pro), trans-3-hydroxyproline (3-Hyp) or cis-4-hydroxyproline (4-hyp) residue substituting for 2,3-trans-3,4-trans-dihydroxyproline in the natural product. We report herein an efficient strategy that provides a dipeptide containing a (2S,4S)-4,5-dihydroxyleucine (dihyLeu) residue, including a diastereoselective dihydroxylation. Nα-Carbobenzyloxy-(2S)-4,5-dehydroleucine was coupled with valine ethyl ester to give a dipeptide that was subjected to a Sharpless asymmetric dihydroxylation to introduce the diol. The relative configuration at C4 was assigned as S by X-ray crystallography after derivatization as an α-amino-γ-lactone hydrochloride salt. The preparation of the 2-(methylsulfonyl)tryptophan residue is described followed by incorporation into a tetrapeptide, Fmoc-Ala-[2-MeSO2]-Trp-diHyLeu(OTBS)-Val-OEt. An efficient synthesis of four tripeptide fragments is also described: Fmoc-D-Thr(OTBS)-D-Ser(OTBS)-Pro*-OBn, where Pro* represents Pro, 3-Hyp, 4-hyp and DHP. These tripeptides were assembled via a [2+1] coupling between Fmoc-D-Thr(OtBu)-D-Ser(OtBu)-OH and the appropriate proline benzyl ester. The acid labile side-chain protecting groups were swapped out for fluoride-labile silyl ethers. Linear heptapeptides were prepared via [3+4] fragment condensations between the series of four tripeptide acids Fmoc-D-Thr(OTBS)-D-Ser(OTBS)-Pro*-OH and the tetrapeptide amine H-Ala-[2-MeSO2-Trp]-diHyLeu(OTBS)-Val-OEt. Deprotection of the N- and C-terminii, followed by cyclization and global side chain deprotection generated our target cyclopeptides. Removal of excess TBAF reagent and salts formed as byproducts during ethyl ester and silyl ether deprotections was achieved by treatment with DOWEX 50WX8-400 H+ resin and calcium carbonate. This procedure led to reasonable yields of the three analogs but afforded only trace amounts of the natural product after HPLC purification. We examined the conformational preferences of dipeptide fragments Ac-D-Ser-Pro*-NHMe (in both free and TBS protected side chains of D-Ser and Pro* residues) using computational studies. The computational analyses confirm that the ratio of trans:cis conformers varies with the degree, regio- and stereochemistry of proline hydroxylation. These equilibrium constant about the prolyl amide bond calculated for these dipeptides are in qualitative agreement with those determined by NMR for tripeptides Fmoc-D-Thr-D-Ser-Pro*-OBn (in both free and TBS protected side chains)

Cell-Penetrating Peptides: design strategies beyond primary structure and amphipathicity

Efficient intracellular drug delivery and target specificity are often hampered by the presence of biological barriers. Thus, compounds that efficiently cross cell membranes are the key to improving the therapeutic value and on-target specificity of non-permeable drugs. The discovery of cell-penetrating peptides (CPPs) and the early design approaches through mimicking the natural penetration domains used by viruses have led to greater efficiency of intracellular delivery. Following these nature-inspired examples, a number of rationally designed CPPs has been developed. In this review, a variety of CPP designs will be described, including linear and flexible, positively charged and often amphipathic CPPs, and more rigid versions comprising cyclic, stapled, or dimeric and/or multivalent, self-assembled peptides or peptido-mimetics. The application of distinct design strategies to known physico-chemical properties of CPPs offers the opportunity to improve their penetration efficiency and/or internalization kinetics. This led to increased design complexity of new CPPs that does not always result in greater CPP activity. Therefore, the transition of CPPs to a clinical setting remains a challenge also due to the concomitant involvement of various internalization routes and heterogeneity of cells used in the in vitro studies