Raymond Behrendt

Ultrafast spectroscopy reveals subnanosecond peptide conformational dynamics and validates molecular dynamics simulation.

Femtosecond time-resolved spectroscopy on model peptides with built-in light switches combined with computer simulation of light-triggered motions offers an attractive integrated approach toward the understanding of peptide conformational dynamics. It was applied to monitor the light-induced relaxation dynamics occurring on subnanosecond time scales in a peptide that was backbone-cyclized with an azobenzene derivative as optical switch and spectroscopic probe. The femtosecond spectra permit the clear distinguishing and characterization of the subpicosecond photoisomerization of the chromophore, the subsequent dissipation of vibrational energy, and the subnanosecond conformational relaxation of the peptide. The photochemical cis/trans-isomerization of the chromophore and the resulting peptide relaxations have been simulated with molecular dynamics calculations. The calculated reaction kinetics, as monitored by the energy content of the peptide, were found to match the spectroscopic data. Thus we verify that all-atom molecular dynamics simulations can quantitatively describe the subnanosecond conformational dynamics of peptides, strengthening confidence in corresponding predictions for longer time scales.

Picosecond conformational transition and equilibration of a cyclic peptide

Ultrafast IR spectroscopy is used to monitor the nonequilibrium backbone dynamics of a cyclic peptide in the amide I vibrational range with picosecond time resolution. A conformational change is induced by means of a photoswitch integrated into the peptide backbone. Although the main conformational change of the backbone is completed after only 20 ps, the subsequent equilibration in the new region of conformational space continues for times >16 ns. Relaxation and equilibration processes of the peptide backbone occur on a discrete hierarchy of time scales. Albeit possessing only a few conformational degrees of freedom compared with a protein, the peptide behaves highly nontrivially and provides insights into the complexity of fast protein folding.

Mammalian RNase H2 removes ribonucleotides from DNA to maintain genome integrity

Mouse RNase H2 is essential to remove ribonucleotides from the genome to prevent DNA damage.

Photomodulation of the Conformation of Cyclic Peptides with Azobenzene Moieties in the Peptide Backbone

The cisright harpoon over left harpoon trans photoisomerization of the azobenzene building block 4-(4-aminophenylazo)benzoic acid incorporated in a cyclic peptide (see scheme) facilitated a two-state transition of the peptide chain from a rigid constrained conformation in the trans isomer into the largely free conformational space of the cis isomer.

Mouse SAMHD1 Has Antiretroviral Activity and Suppresses a Spontaneous Cell-Intrinsic Antiviral Response

SUMMARY Aicardi-Goutières syndrome (AGS), a hereditary autoimmune disease, clinically and biochemically overlaps with systemic lupus erythematosus (SLE) and, like SLE, is characterized by spontaneous type I interferon (IFN) production. The finding that defects of intracellular nucleases cause AGS led to the concept that intracellular accumulation of nucleic acids triggers inappropriate production of type I IFN and autoimmunity. AGS can also be caused by defects of SAMHD1, a 3′ exonuclease and deoxy-nucleotide (dNTP) triphosphohydrolase. Human SAMHD1 is an HIV-1 restriction factor that hydrolyzes dNTPs and decreases their concentration below the levels required for retroviral reverse transcription. We show in gene-targeted mice that also mouse SAMHD1 reduces cellular dNTP concentrations and restricts retroviral replication in lymphocytes, macrophages, and dendritic cells. Importantly, the absence of SAMHD1 triggered IFN-β-dependent transcriptional upregulation of type I IFN-inducible genes in various cell types indicative of spontaneous IFN production. SAMHD1-deficient mice may be instrumental for elucidating the mechanisms that trigger pathogenic type I IFN responses in AGS and SLE.

Photomodulation of conformational states. Synthesis of cyclic peptides with backbone-azobenzene moieties.

The search for photoresponsive conformational transitions accompanied by changes in physicochemical and biological properties led us to the design of small cyclic peptides containing azobenzene moieties in the backbone. For this purpose, (4‐aminomethyl)phenylazobenzoic acid (H‐AMPB‐OH) and (4‐amino)phenylazobenzoic acid (H‐APB‐OH) were synthesized and used to cyclize a bis‐cysteinyl‐octapeptide giving monocyclic derivatives in which additional conformational restriction could be introduced by conversion to bicyclic structures with a disulphide bridge. While synthesis with H‐AMPB‐OH proceeded smoothly on a chlorotrityl‐resin with Fmoc/tBu chemistry, the poor nucleophilicity of the arylamino group of H‐APB‐OH required special chemistry for satisfactory incorporation into the peptide chain. Additional difficulties were encountered in the reductive cleavage of the S‐tert‐butylthio group from the cysteine residues since concomitant reduction of the azobenzene moiety took place at competing rates. This difficulty was eventually bypassed by using the S‐trityl protection. Side‐chain cyclization of the APB‐peptide proved to be difficult, suggesting that restricted conformational freedom was already present in the monocyclic form, a fact that was fully confirmed by NMR structural analysis. Conversely, the methylene spacer in the AMPB moiety introduced sufficient flexibility for facile and quantitative side‐chain cyclization to the bicyclic form. Both of the monocyclic peptides and both of the bicyclic peptides are photoresponsive molecules which undergo cis/trans isomerization reversibly. Copyright

Ultrafast Conformational Dynamics in Cyclic Azobenzene Peptides of Increased Flexibility

Structural changes of peptides containing the azobenzene dye 4-aminomethyl-phenylazobenzoic acid (AMPB) are studied with ultrafast spectroscopy. AMPB peptides are a new class of molecules where the photoisomerizable dye azobenzene is linked to the peptide moiety via a flexible methylene spacer. The ultrafast reactions in the femtosecond to nanosecond time domain are investigated for the optical switch AMPB, a linear and cyclic octapeptide, and a bicyclic octapeptide containing an additional disulfide bridge. These molecules with increasing conformational constraints are studied for the cis to trans and the trans to cis photoreactions. For the cis to trans reaction the isomerization of the chromophore occurs fast in the 1-ps range, whereas it is slower (10-ps range) in the trans to cis reaction. In all peptides the structural changes of the chromophore lead to modifications in the peptide structure in the 10-ps-1-ns time range. The results indicate that the chromophore AMPB acts simultaneously as a fast molecular switch and as a sensor for initial conformational dynamics in the peptide. Experiments in the mid-infrared range where the structural changes of the peptide backbone are directly observed demonstrate that the essential part of the structural dynamics in the bicyclic AMPB peptide occurs faster than 10 ns.

Photomodulation of conformational states. I. Mono- and bicyclic peptides with (4-amino)phenylazobenzoic acid as backbone constituent†

The thioredoxin reductase active-site fragment H-Ala-Cys-Ala-Thr-Cys-Asp-Gly-Phe-OH [134-141], which is known for its high tendency to assume an almost identical conformation as in the intact enzyme, was backbone cyclized with the photoresponsive (4-amino)phenylazobenzoic acid (APB) to produce a monocyclic and disulfide-bridged bicyclic APB-peptide. Light-induced reversible cis/trans isomerization occurs at identical extents in both the linear and the two cyclic forms. Nuclear magnetic resonance conformational analysis clearly revealed that in the bicyclic APB-peptide both as a trans- and cis-azo-isomer the constraints imparted by the bicyclic structure do not allow the molecule to relax into a defined low energy conformation, thus making the molecule a frustrated system that flip-flops between multiple conformational states. Conversely, the monocyclic APB peptide folds into a well-defined lowest energy structure as a trans-azo-isomer, which upon photoisomerization to the cis-azo configuration relaxes into a less restricted conformational space. First femtosecond spectroscopic analysis of the dynamics of the photoreaction confirm a fast first phase on the femtosecond time scale related to the cis/trans isomerization of the azobenzene moiety followed by a slower phase in the picosecond time scale that involves an adjustment of the peptide backbone. Due to the well- defined photoresponsive two-state transition of this monocyclic peptide molecule, it represents a model system well suited for studying the ultrafast dynamics of conformational transitions by time-resolved spectroscopy.

Photomodulation of conformational states. II. Mono‐ and bicyclic peptides with (4‐aminomethyl)phenylazobenzoic acid as backbone constituent

It has been reported that backbone cyclization of octapeptides with the photoresponsive (4-aminomethyl)phenylazobenzoic acid imparts sufficient restraints to induce and stabilize ordered conformations of the peptide backbone in both the cis- and trans-azo-isomers (L. Ulysse, J. Cubillos, and J. Chmielewski, Journal of the American Chemical Society, 1995, Vol. 117, pp. 8466-8467). Correspondingly, the active-site octapeptide fragment H-Ala-Cys-Ala-Thr-Cys-Asp-Gly-Phe-OH [134-141] of thioredoxin reductase, with its high preference for a 3(10)-helix turn conformation centered on the Thr-Cys sequence, was backbone cyclized with this azobenzene moiety in the attempt to design a photoresponsive system where the conformational states of the peptide backbone are dictated by the configuration of the azobenzene and can be further modulated by the disulfide bridge. Nuclear magnetic resonance conformational analysis of the monocyclic compound clearly revealed the presence of two conformational families in both the cis- and trans-azo configuration. Of the higher populated conformational families, the structure of the trans-isomer seems like a pretzel-like folding, while the cis-isomer relaxes into a significantly less defined conformational state that does not exhibit any regular structural elements. Further restrictions imparted by disulfide bridging of the peptide moiety leads to an even better defined conformation for the trans-azo-isomer, whereas the cis-isomer can be described as a frustrated system without pronounced energy minima and thus with little conformational preferences. Our findings would suggest that this photoresponsive peptide template may not be of general usefulness for light-induced conformational transitions between two well-defined conformational states at least under the experimental conditions employed, even in the bicyclic form. However, trans --> cis isomerization of the bicyclic peptide is accompanied by a switch from a well-defined conformation to an ensemble of possible conformations.

PHOTOMODULIERUNG DER KONFORMATION CYCLISCHER PEPTIDE MIT AZOBENZOL-BAUSTEINEN IM PEPTIDRUCKGRAT

Diecis⇌trans-Photoisomerisierung des in ein cyclisches Peptid eingebauten Azobenzol-Bausteins 4-(4-Aminophenylazo)benzoesaure (siehe Schema) ermoglicht die Lichtschaltung eines Zwei-Zustande-Ubergangs von einer starren, konformativ eingeschrankten Struktur des trans-Isomers in den weitgehend freien Konformationsraum der cis-Form.