Inversion of the Side‐Chain Stereochemistry of Indvidual Thr or Ile Residues in a Protein Molecule: Impact on the Folding,Stability, and Structure of the ShK Toxin

ShK toxin is a cysteine‐rich 35‐residue protein ion‐channel ligand isolated from the sea anemone Stichodactyla helianthus. In this work, we studied the effect of inverting the side chain stereochemistry of individual Thr or Ile residues on the properties of the ShK protein. Molecular dynamics simulations were used to calculate the free energy cost of inverting the side‐chain stereochemistry of individual Thr or Ile residues. Guided by the computational results, we used chemical protein synthesis to prepare three ShK polypeptide chain analogues, each containing either an allo‐Thr or an allo‐Ile residue. The three allo‐Thr or allo‐Ile‐containing ShK polypeptides were able to fold into defined protein products, but with different folding propensities. Their relative thermal stabilities were measured and were consistent with the MD simulation data. Structures of the three ShK analogue proteins were determined by quasi‐racemic X‐ray crystallography and were similar to wild‐type ShK. All three ShK analogues retained ion‐channel blocking activity.     

Affinity‐Labeling‐Based Introduction of a Reactive Handle for Natural Protein Modification

A new chemical method to site‐specifically modify natural proteins without the need for genetic manipulation is described. Our strategy involves the affinity‐labeling‐based attachment of a unique reactive handle at the surface of the target protein, and the subsequent selective transformation of the reactive handle by a bioorthogonal reaction to introduce a variety of functional probes into the protein. To demonstrate this approach, we synthesized labeling reagents that contain: 1) a benzenesulfonamide ligand that directs specifically to bovine carbonic anhydrase II (bCA), 2) an electrophilic epoxide group for protein labeling, 3) an exchangeable hydrazone bond linking the ligand and the epoxide group, and 4) an iodophenyl or acetylene handle. By incubating the labeling reagent with bCA, the reactive handle was covalently attached at the surface of bCA through epoxide ring opening. Either after or before removing the ligand by a hydrazone/oxime‐exhange reaction, which restores the enzymatic activity, the reactive handle incorporated could be derivatized by Suzuki coupling or Huisgen cycloaddition reactions. This method is also applicable to the target‐specific multiple modification in a protein mixture. The availability of various (photo)affinity‐labeling reagents and bioorthogonal reactions should extend the flexibility of this strategy for the site‐selective incorporation of many functional molecules into proteins.     

Enzymatic Control of the Conformational Landscape of Self‐Assembling Peptides

Post‐translational modification is a common mechanism to affect conformational change in proteins, which in turn, regulates function. Herein, this principle is expanded to instruct the formation of supramolecular assemblies by controlling the conformational bias of self‐assembling peptides. Biophysical and mechanical studies show that an engineered phosphorylation/dephosphorylation couple can affectively modulate the folding of amphiphilic peptides into a conformation necessary for the formation of well‐defined fibrillar networks. Negative design principles based on the incompatibility of hosting residue side‐chain point charge within hydrophobic environments proved key to inhibiting the peptide's ability to adopt its low energy fold in the assembled state. Dephosphorylation relieves this restriction, lowers the energy barrier between unfolded and folded peptide, and allows the formation of self‐assembled fibrils that contain the folded conformer, thus ultimately enabling the formation of a cytocompatible hydrogel material.     

MetREx: A protein design approach for the exploration of sequence‐reactivity relationships in metalloenzymes

Metalloenzymes represent a particular challenge for any rational (re)design approach because the modeling of reaction events at their metallic cofactors requires time‐consuming quantum mechanical calculations, which cannot easily be reconciled with the fast, knowledge‐based approaches commonly applied in protein design studies. Here, an approach for the exploration of sequence‐reactivity relationships in metalloenzymes is presented (MetREx) that consists of force field‐based screening of mutants that lie energetically between a wild‐type sequence and the global minimum energy conformation and which should, therefore, be compatible with a given protein fold. Mutant candidates are subsequently evaluated with a fast and approximate quantum mechanical/molecular mechanical‐like procedure that models the influence of the protein environment on the active site by taking partial charges and van der Waals repulsions into account. The feasibility of the procedure is demonstrated for the active site of [FeFe] hydrogenase from Desulfovibrio desulfuricans. The method described allows for the identification of mutants with altered properties, such as inhibitor‐coordination energies, and the understanding of the robustness of enzymatic reaction steps with respect to variations in sequence space. © 2015 Wiley Periodicals, Inc.     

Rapid Activation of Platinum with Black Phosphorus for Efficient Hydrogen Evolution

Modulation of the electronic structure of metal catalysts is an effective approach to optimize the electrocatalytic activity. Herein, we show a surprisingly strong activation effect of black phosphorus (BP) on platinum (Pt) catalysts to give greatly enhanced catalytic activity in the hydrogen evolution reaction (HER). The unique and negative binding energy between BP and Pt leads to spontaneous formation of Pt‐P bonds producing strong synergistic ligand effects on the Pt nanoparticles. No Pt‐P bonds are formed with red phosphorus which is another allotrope of P. By controlling the number of Pt‐P bonds, 3.5‐fold enhancement in the HER activity can be achieved from the BP‐activated Pt catalyst and the activity is 6.1 times higher than that of the state‐of‐the‐art commercial Pt/C catalyst. The BP‐activated Pt catalyst exhibits a current density of 82.89 mA cm^?2 with only 1 μg of Pt in 1 m KOH at an overpotential of 70 mV.     

Structure‐Based Design of a Macrocyclic PROTAC

Constraining a molecule in its bioactive conformation via macrocyclization represents an attractive strategy to rationally design functional chemical probes. While this approach has been applied to enzyme inhibitors or receptor antagonists, to date it remains unprecedented for bifunctional molecules that bring proteins together, such as PROTAC degraders. Herein, we report the design and synthesis of a macrocyclic PROTAC by adding a cyclizing linker to the BET degrader MZ1. A co‐crystal structure of macroPROTAC‐1 bound in a ternary complex with VHL and the second bromodomain of Brd4 validated the rational design. Biophysical studies revealed enhanced discrimination between the second and the first bromodomains of BET proteins. Despite a 12‐fold loss of binary binding affinity for Brd4, macroPROTAC‐1 exhibited cellular activity comparable to MZ1. Our findings support macrocyclization as an advantageous strategy to enhance PROTAC degradation potency and selectivity between homologous targets.     

Radiation Damage and Racemic Protein Crystallography Reveal the Unique Structure of the GASA/Snakin Protein Superfamily

Proteins from the GASA/snakin superfamily are common in plant proteomes and have diverse functions, including hormonal crosstalk, development, and defense. One 63‐residue member of this family, snakin‐1, an antimicrobial protein from potatoes, has previously been chemically synthesized in a fully active form. Herein the 1.5 Å structure of snakin‐1, determined by a novel combination of racemic protein crystallization and radiation‐damage‐induced phasing (RIP), is reported. Racemic crystals of snakin‐1 and quasi‐racemic crystals incorporating an unnatural 4‐iodophenylalanine residue were prepared from chemically synthesized d ‐ and l ‐proteins. Breakage of the C?I bonds in the quasi‐racemic crystals facilitated structure determination by RIP. The crystal structure reveals a unique protein fold with six disulfide crosslinks, presenting a distinct electrostatic surface that may target the protein to microbial cell surfaces.     

Computational design of a single amino acid sequence that can switch between two distinct protein folds

The functions of many proteins are mediated by specific conformational changes, and therefore the ability to design primary sequences capable of secondary and tertiary changes is an important step toward the creation of novel functional proteins. To this end, we have developed an algorithm that can optimize a single amino acid sequence for multiple target structures. The algorithm consists of an outer loop, in which sequence space is sampled by a Monte Carlo search with simulated annealing, and an inner loop, in which the effect of a given mutation is evaluated on the various target structures by using the rotamer packing routine and composite energy function of the protein design software, RosettaDesign. We have experimentally tested the method by designing a peptide, Sw2, which can be switched from a 2Cys-2His zinc finger-like fold to a trimeric coiled-coil fold, depending upon the pH or the presence of transition metals. Physical characterization of Sw2 confirms that it is able to reversibly adopt each intended target fold.     

Folding a nonbiological polymer into a compact multihelical structure

The only molecules that are currently known to fold into unique three-dimensional conformations and perform sophisticated functions are biological polymers - proteins and some RNA molecules. Our aim is to create a nonbiological sequence-specific polymer that folds in aqueous solution. Toward that end, we synthesized sequence-specific 30mer, 45mer, and 60mer peptoid oligomers (N-substituted glycine polymers) consisting of 15mer units we chained together by disulfide and oxime linkages to mimic the helical bundle structures commonly found in proteins. Because these 15mer sequences were previously shown to form defined helical structures that aggregate together at submillimolar concentrations, we expected that by covalently linking multiple 15mers together, they might fold as helical bundles. To probe whether they folded, we used fluorescence resonance energy transfer (FRET) reporter groups. We found that certain constructs fold up with a hydrophobic core and have cooperative folding transitions. Such molecules may ultimately provide a platform for designing specific functions resembling those of proteins.     

Rethinking Cysteine Protective Groups: S‐Alkylsulfonyl‐l‐Cysteines for Chemoselective Disulfide Formation

The ability to reversibly cross‐link proteins and peptides grants the amino acid cysteine its unique role in nature as well as in peptide chemistry. We report a novel class of S‐alkylsulfonyl‐l ‐cysteines and N‐carboxy anhydrides (NCA) thereof for peptide synthesis. The S‐alkylsulfonyl group is stable against amines and thus enables its use under Fmoc chemistry conditions and the controlled polymerization of the corresponding NCAs yielding well‐defined homo‐ as well as block co‐polymers. Yet, thiols react immediately with the S‐alkylsulfonyl group forming asymmetric disulfides. Therefore, we introduce the first reactive cysteine derivative for efficient and chemoselective disulfide formation in synthetic polypeptides, thus bypassing additional protective group cleavage steps.     

A Naturally Encoded Dipeptide Handle for Bioorthogonal Chan–Lam Coupling

Manipulation of biomacromolecules is ideally achieved through unique and bioorthogonal chemical reactions of genetically encoded, naturally occurring functional groups. The toolkit of methods for site‐specific conjugation is limited by selectivity concerns and a dearth of naturally occurring functional groups with orthogonal reactivity. We report that pyroglutamate amide N?H bonds exhibit bioorthogonal copper‐catalyzed Chan–Lam coupling at pyroglutamate‐histidine dipeptide sequences. The pyroglutamate residue is readily incorporated into proteins of interest by natural enzymatic pathways, allowing specific bioconjugation at a minimalist dipeptide tag.     

Optische Speichermedien. Phasenwechselmaterialien

The information in DVDs and Blu‐Ray Disks is stored in a thin layer of tellurium based materials. Using short Laser pulses with appropriate energy the material can be reversibly switched between the crystalline and amorphous states. The two states exhibit a pronounced optical and electrical contrast making this class of materials – often termed as phase change materials – unique.     

Structure of Human Tyrosinase Related Protein 1 Reveals a Binuclear Zinc Active Site Important for Melanogenesis

Tyrosinase‐related protein 1 (TYRP1) is one of three tyrosinase‐like glycoenzymes in human melanocytes that are key to the production of melanin, the compound responsible for the pigmentation of skin, eye, and hair. Difficulties with producing these enzymes in pure form have hampered the understanding of their activity and the effect of mutations that cause albinism and pigmentation disorders. Herein we show that the typical tyrosinase‐like subdomain of TYRP1 contains two zinc ions in the active site instead of copper ions as found in tyrosinases, which explains why TYRP1 does not exhibit tyrosinase redox activity. In addition, the structures reveal for the first time that the Cys‐rich subdomain, which is unique to vertebrate melanogenic proteins, has an epidermal growth factor‐like fold and is tightly associated with the tyrosinase subdomain. Our structures suggest that most albinism‐related mutations of TYRP1 affect its stability or activity.     

Catalysis and Electron Transfer in De Novo Designed Helical Scaffolds

The relationship between protein structure and function is one of the greatest puzzles within biochemistry. De novo metalloprotein design is a way to wipe the board clean and determine what is required to build in function from the ground up in an unrelated structure. This Review focuses on protein design efforts to create de novo metalloproteins within alpha‐helical scaffolds. Examples of successful designs include those with carbonic anhydrase or nitrite reductase activity by incorporating a ZnHis₃ or CuHis₃ site, or that recapitulate the spectroscopic properties of unique electron‐transfer sites in cupredoxins (CuHis₂Cys) or rubredoxins (FeCys₄). This work showcases the versatility of alpha helices as scaffolds for metalloprotein design and the progress that is possible through careful rational design. Our studies cover the invariance of carbonic anhydrase activity with different site positions and scaffolds, refinement of our cupredoxin models, and enhancement of nitrite reductase activity up to 1000‐fold.     

Inversion of the Side‐Chain Stereochemistry of Indvidual Thr or Ile Residues in a Protein Molecule: Impact on the Folding,Stability, and Structure of the ShK Toxin

ShK toxin is a cysteine‐rich 35‐residue protein ion‐channel ligand isolated from the sea anemone Stichodactyla helianthus. In this work, we studied the effect of inverting the side chain stereochemistry of individual Thr or Ile residues on the properties of the ShK protein. Molecular dynamics simulations were used to calculate the free energy cost of inverting the side‐chain stereochemistry of individual Thr or Ile residues. Guided by the computational results, we used chemical protein synthesis to prepare three ShK polypeptide chain analogues, each containing either an allo‐Thr or an allo‐Ile residue. The three allo‐Thr or allo‐Ile‐containing ShK polypeptides were able to fold into defined protein products, but with different folding propensities. Their relative thermal stabilities were measured and were consistent with the MD simulation data. Structures of the three ShK analogue proteins were determined by quasi‐racemic X‐ray crystallography and were similar to wild‐type ShK. All three ShK analogues retained ion‐channel blocking activity.     

Exploring the parameter space of the coarse‐grained UNRES force field by random search: Selecting a transferable medium‐resolution force field

We explored the energy‐parameter space of our coarse‐grained UNRES force field for large‐scale ab initio simulations of protein folding, to obtain good initial approximations for hierarchical optimization of the force field with new virtual‐bond‐angle bending and side‐chain‐rotamer potentials which we recently introduced to replace the statistical potentials. 100 sets of energy‐term weights were generated randomly, and good sets were selected by carrying out replica‐exchange molecular dynamics simulations of two peptides with a minimal α‐helical and a minimal β‐hairpin fold, respectively: the tryptophan cage (PDB code: 1L2Y) and tryptophan zipper (PDB code: 1LE1). Eight sets of parameters produced native‐like structures of these two peptides. These eight sets were tested on two larger proteins: the engrailed homeodomain (PDB code: 1ENH) and FBP WW domain (PDB code: 1E0L); two sets were found to produce native‐like conformations of these proteins. These two sets were tested further on a larger set of nine proteins with α or α + β structure and found to locate native‐like structures of most of them. These results demonstrate that, in addition to finding reasonable initial starting points for optimization, an extensive search of parameter space is a powerful method to produce a transferable force field. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2009     

Dynamic Modulation of Enzyme Activity by Near‐Infrared Light

Engineering near‐infrared (NIR) light‐sensitive enzymes remains a huge challenge. A photothermal effect‐associated method is developed for tailoring the enzymatic activity of enzymes by exposure to NIR light. An ultrasmall platinum nanoparticle was anchored in an enzyme to generate local heating upon NIR irradiation, which enhanced the enzyme activity without increasing bulk temperature. Following NIR irradiation, the enzyme activity was tailored rapidly and reversibly, and was modulated by varying laser power density and irradiation time. Four enzymes were engineered, including glucoamylase, glucose oxidase, catalase, and proteinase K with NIR‐light sensitivity, and demonstrated their utility in practical applications such as photolithography and NIR light‐responsive antibacterial or anticancer actions. Our investigation suggests that this approach could be broadly used to engineer enzymes with NIR‐light sensitivity for many biological applications.     

New energy terms for reduced protein models implemented in an off‐lattice force field

Parameterization and test calculations of a reduced protein model with new energy terms are presented. The new energy terms retain the steric properties and the most significant degrees of freedom of protein side chains in an efficient way using only one to three virtual atoms per amino acid residue. The energy terms are implemented in a force field containing predefined secondary structure elements as constraints, electrostatic interaction terms, and a solvent‐accessible surface area term to include the effect of solvation. In the force field the main‐chain peptide units are modeled as electric dipoles, which have constant directions in α‐helices and β‐sheets and variable conformation‐dependent directions in loops. Protein secondary structures can be readily modeled using these dipole terms. Parameters of the force field were derived using a large set of experimental protein structures and refined by minimizing RMS errors between the experimental structures and structures generated using molecular dynamics simulations. The final average RMS error was 3.7 Å for the main‐chain virtual atoms (C_α atoms) and 4.2 Å for all virtual atoms for a test set of 10 proteins with 58–294 amino acid residues. The force field was further tested with a substantially larger test set of 608 proteins yielding somewhat lower accuracy. The fold recognition capabilities of the force field were also evaluated using a set of 27,814 misfolded decoy structures. © 2001 John Wiley & Sons, Inc. J Comput Chem 22: 1229–1242, 2001     

Surfactant‐Stabilized Small Hydrogel Particles in Oil: Hosts for Remarkable Activation of Enzymes in Organic Solvents

Hydrogels of amino acid based cationic surfactant having C₁₆ tails were used to immobilize heme proteins and enzyme. These hydrogel‐entrapped proteins/enzyme showed remarkable activation when dispersed in organic solvent. The activation effect (ratio of the activity of the hydrogel‐entrapped enzyme in organic solvent to the activity of the native enzyme in water) of cytochrome c increased up to 350‐fold with varying protein and gelator concentration. Hydrogel‐entrapped hemoglobin and horseradish peroxidase (HRP) also showed markedly improved activity in organic solvent. Alteration in the structure of the gelator and its supramolecular arrangement showed that the protein immobilized within amphiphilic networks with larger interstitial space exhibited higher activation. This striking activation of hydrogel‐entrapped proteins stems from the following effects: 1) the hydrophilic domain of the amphiphilic networks facilitates accessibility of the enzyme to the water‐soluble substrate. 2) the surfactant, as an integral part of the amphiphilic network, assists in the formation of a distinct interface through which reactants and products are easily transferred between hydrophilic and hydrophobic domains. 3) Surfactant gelators help in the dispersion and stabilization of gel matrix into small particles in organic solvent, which enhances the overall surface area and results in improved mass transfer. The activation was dramatically improved up to 675‐fold in the presence of nongelating anionic surfactants that helped in disintegration of the gel into further smaller‐sized particles. Interestingly, hydrogel‐immobilized HRP exhibited about 2000‐fold higher activity in comparison to the activity of the suspended enzyme in toluene. Structural changes of the entrapped enzyme and the morphology of the matrix were investigated to understand the mechanism of this activation.     

Impaired Chaperone Activity of Human Heat Shock Protein Hsp27 Site‐Specifically Modified with Argpyrimidine

Non‐enzymatic posttranslational modifications (nPTMs) affect at least ～30 % of human proteins, but our understanding of their impact on protein structure and function is limited. Studies of nPTMs are difficult because many modifications are not included in common chemical libraries or protein expression systems and should be introduced site‐specifically. Herein, we probed the effect of the nPTM argpyrimidine on the structure and function of human protein Hsp27, which acquires argpyrimidine at residue 188 in vivo. We developed a synthetic approach to an argpyrimidine building block, which we then incorporated at position 188 of Hsp27 through protein semisynthesis. This modification did not affect the protein secondary structure, but perturbed the oligomeric assembly and impaired chaperone activity. Our work demonstrates that protein function can be altered by a single nPTM and opens up a new area of investigation only accessible by methods that allow site‐selective protein modification.