Applying Peptide and Protein Synthesis to Study Post-translational Modifications in Epigenetics and Beyond

Epigenetics research focuses on the study of heritable gene regulatory mechanisms that do not involve changes of the DNA sequence. Such mechanisms include post-translational modifications of histone proteins that organize the genome in the nucleus into a nucleoprotein complex called chromatin, and which are of key importance in development and disease. Chemical biology tools as developed by my group, in particular synthetic peptide and protein chemistry, have been critical to elucidate epigenetic signaling mechanisms. As outlined below, they allow the reconstitution of chromatin carrying defined modifications and thus the elucidation of detailed molecular mechanisms

Testing the diffusing boundary model for the helix-coil transition in peptides

The dynamics of peptide α-helices have been studied extensively for many years, and the kinetic mechanism of the helix-coil dynamics has been discussed controversially. Recent experimental results have suggested that equilibrium helix-coil dynamics are governed by movement of the helix/coil boundary along the peptide chain, which leads to slower unfolding kinetics in the helix center compared with the helix ends and position-independent helix formation kinetics. We tested this diffusion of boundary model in helical peptides of different lengths by triplet-triplet energy transfer measurements and compared the data with simulations based on a kinetic linear Ising model. The results show that boundary diffusion in helical peptides can be described by a classical, Einstein-type, 1D diffusion process with a diffusion coefficient of 2.7⋅107 (amino acids)2/s or 6.1⋅10-9 cm2/s. In helices with a length longer than about 40 aa, helix unfolding by coil nucleation in a helical region occurs frequently in addition to boundary diffusion. Boundary diffusion is slowed down by helix-stabilizing capping motifs at the helix ends in agreement with predictions from the kinetic linear Ising model. We further tested local and nonlocal effects of amino acid replacements on helix-coil dynamics. Single amino acid replacements locally affect folding and unfolding dynamics with a f-value of 0.35, which shows that interactions leading to different helix propensities for different amino acids are already partially present in the transition state for helix formation. Nonlocal effects of amino acid replacements only influence helix unfolding (f = 0) in agreement with a diffusing boundary mechanism

Histone H2B ubiquitylation disrupts local and higher-order chromatin compaction

Regulation of chromatin structure involves histone posttranslational modifications that can modulate intrinsic properties of the chromatin fiber to change the chromatin state. We used chemically defined nucleosome arrays to demonstrate that H2B ubiquitylation (uH2B), a modification associated with transcription, interferes with chromatin compaction and leads to an open and biochemically accessible fiber conformation. Notably, these effects were specific for ubiquitin, as compaction of chromatin modified with a similar ubiquitin-sized protein, Hub1, was only weakly affected. Applying a fluorescence-based method, we found that uH2B acts through a mechanism distinct from H4 tail acetylation, a modification known to disrupt chromatin folding. Finally, incorporation of both uH2B and acetylated H4 resulted in synergistic inhibition of higher-order chromatin structure formation, possibly a result of their distinct modes of action

Dynamics of unfolded and [alpha]-helical polypeptide chains

The aim of this thesis was the investigation of the dynamics of elementary steps in protein folding. During folding, the polypeptide chain explores the free energy surface and first interactions are established. These interactions lead to formation of secondary structure elements which then can assemble to form protein structure. Contact formation between different residues in the polypeptide chain limits the rate with which a protein can explore its conformational space and sets an upper limit for the speed of folding. We studied loop closure reactions in polypeptides and dynamics of α-helices applying the method of triplet-triplet energy transfer (TTET). Triplet excitation is transferred by a two electron exchange mechanism from a xanthone (Xan) donor moiety to a naphthalene (Nal) acceptor upon van der Waals contact. The transfer reaction is diffusion controlled allowing direct determination of rate constants for loop formation by observing the decay of xanthone triplet absorption or the concomitant increase in naphthalene triplets. By introducing the triplet labels into unfolded model peptides we wanted to test the effect of polypeptide chain length, amino acid sequence and solvent conditions. In a short host-guest loop Xan-Ser-Xaa-Ser-Nal-Ser-Gly-OH the local effect of different amino acids on loop formation was probed by introducing the guest amino acids Xaa = Ser, Gly, Pro, Ala, Ile, Glu, Arg and His. We observed similar kinetics for all amino acids except glycine and proline, although amino acids with a Cβ-atom showed slightly slower loop closure rate constants. In glycine containing peptides the contact formation rate constant was found to be faster because of the increased flexibility of the glycine residue. The introduction of proline however led to double exponential kinetics. A slow phase corresponding to trans-Pro showed the slowest kinetics of all peptides due to the rigid structure of the proline residue, whereas cis-Pro showed the fastest kinetics due to the introduction of a kink and thus smaller end-toend distances. To probe chain dynamics in a natural loop derived from a protein we introduced the TTET labels into an unstructured 18-residue loop from carp muscle β-parvalbumin. This allowed us to compare loop formation rate constants obtained in a natural sequence to values obtained in model peptides. The kinetics in the parvalbumin loop corresponded well to values obtained for poly-Ser chains. This showed that a small amount of glycine in the sequence can compensate for the slowing down of chain dynamics induced by large amino acids. During protein folding, interactions are mostly established between amino acids in the interior of the chain. Thus, the effect of additional tails on the loop closure rate constants has to be taken into account. Three types of loops can be distinguished. Type I loops denote end-to-end loops, type II-loops are end-to-interior loops, while type III-loops denote interior-to-interior loops. We measured loop formation rate constants in type II and type III-loops depending on the size of the additional tails. It was observed that the loop formation rate constant is decreased with increasing size of the tail. For different type II-loops this effect was found to reach a limit when the tail dimensions are about three times larger than the loop size. In this limit, loop closure rates are decreased by a factor of 2.3. For type III-loops the effect of additional tails was found to be stronger than in the type II case. However, the limiting value could not be determined. Assuming that the limit for type III-loop formation is reached at the same tail size as in the type II case this would result in a decrease in loop closure rates by a factor of four. All observed rate constants in TTET experiments were found to be on the nanosecond time scale. However, faster reaction could not be ruled out. To study reactions on a shorter time scale using TTET it is necessary to understand the photophysics of xanthone triplet formation and energy transfer to naphthalene in detail. Thus, femtosecond timescale experiments were performed to determine the timescale of xanthone intersystem crossing and triplet-triplet energy transfer. It was found that xanthone triplet formation proceeds on the 2 ps timescale and TTET to naphthalene occurs below 2 ps. This allows to observe contact formation processes with time constants of 5-10 ps. As analysis of initial amplitudes in TTET experiments suggested fast reactions that cannot be observed in nanosecond time resolution experiments, we studied contact formation in small peptides applying femtosecond laserflash spectroscopy. Two fast processes were detected. A faster decay with a time constant of 3-4 ps and 15% amplitude was followed by a slower process on the 100’s of picosecond time scale which accounted for 30-40% of the xanthone triplet absorption decay. These fast reactions result from motions of a subpopulation of peptides within a conformational substate on the free energy surface that allows contact without major barrier crossing. The remaining population of molecules has to sample the free energy surface which leads to exponential kinetics in the nanosecond time range at room temperature. To gain more insight into the properties of the free energy surface we tested conditions where barrier crossing between the local minima on the free energy surface is slow. At low temperature or high viscosity the kinetics for loop formation were found to deviate from exponential behaviour and could be described by a stretched exponential decay. These results show that the concept of conformational substates which was initially developed to describe protein motions is valid for unstructured polypeptides. During protein folding, initial contacts lead to formation of local structure. α-helices represent the most abundant and most local secondary structure element. We studied global and local stability and dynamics in α-helices applying TTET to alanine based helical peptides. We introduced TTET labels at different positions and could thus obtain information about local and global helix unfolding and refolding kinetics. We observed that α-helices exhibit higher stability and slower kinetics at central positions, whereas the termini were frayed and showed fluctuations on a faster timescale. These results show that α-helix formation is a complex process and its kinetics are position dependent. To observe elementary reactions of α-helix formation as nucleation steps or propagation reactions, kinetic experiments have to start with an unfolded ensemble. We developed a synthetic access to a peptide system where an α-helical peptide is cyclised by a photocleavable crosslinker moiety based on a p-hydroxyphenacyl moiety which prevents helix formation. This system can now be used to monitor helix formation upon a fast release of the peptide by nanosecond laser irradiation

Dynamic Chromatin Regulation from a Single Molecule Perspective

Chromatin regulatory processes, like all biological reactions, are dynamic and stochastic in nature but can give rise to stable and inheritable changes in gene expression patterns. A molecular understanding of those processes is key for fundamental biological insight into gene regulation, epigenetic inheritance, lineage determination, and therapeutic intervention in the case of disease. In recent years, great progress has been made in identifying important molecular players involved in key chromatin regulatory pathways. Conversely, we, are only beginning to understand the dynamic interplay between protein effectors, transcription factors, and the chromatin substrate itself. Single-molecule approaches employing both highly defined chromatin substrates in vitro, as well as direct observation of complex regulatory processes in vivo, open new avenues for a molecular view of chromatin regulation. This review highlights recent applications of single-molecule methods and related techniques to investigate fundamental chromatin regulatory processes

Revealing chromatin organization in metaphase chromosomes

The local structural organization of chromatin in mitotic chromosomes is not well understood. A new cryo-electron tomography study from the Daban laboratory (Chicano et al, ) reveals that mitotic chromatin isolated from human cells can assume a plate-like fine structure, containing layers of interdigitated nucleosomes. Such a multilayered organization suggests a possible model for mitotic chromatin compaction