Solvent-exposed Position Research Articles

Backbone Modification in a Protein Hydrophobic Core.

Targeted protein backbone modification can recreate tertiary structures reminiscent of folds found in nature on artificial scaffolds with improved biostability. Incorporation of altered monomers in such entities is typically limited to sites distant from the hydrophobic core to avoid potential disruptions to folding. This is limiting, as it is advantageous in some applications to incorporate artificial connectivity at buried sites. Here, we report an examination of protein backbone modification targeted specifically to hydrophobic core positions and its impacts on tertiary folded structure and fold stability. Different artificial monomer types are placed at core, core-flanking, or solvent-exposed positions in a compact three-helix protein. Effects on structure and folding energetics are assessed by NMR spectroscopy and biophysical methods. Results show that artificial residues can be well accommodated in the hydrophobic core of a defined tertiary fold, with effects on stability only modestly larger than identical changes at solvent-exposed sites. Collectively, these results provide new insights into folding behavior of protein-like artificial chains as well as strategies for the design of such molecules.

Comparison of the effect of fluorinated aliphatic and aromatic amino acids on stability at a solvent-exposed position of a globular protein

ABSTRACT Rational protein engineering has often focused on increasing the thermal stability of proteins by incorporating stabilising mutations. Introducing fluorinated amino acids into proteins has been used to enhance protein stability. Free energy simulations of the immunoglobulin binding domain of streptococcal protein G in both the folded and unfolded states were performed to understand the detailed mechanism of the effect of fluorinated aromatic and aliphatic amino acid side chains on the stability of Leu → Hfl (hexafluoroleucine) and Phe → Pff (pentafluorophenylalanine) mutations at the solvent-exposed position of the globular protein. Our simulated free energy differences (ΔΔG) for the Leu → Hfl and Phe → Pff mutations are −0.43 ± 0.14 kcal/mol and −0.45 ± 0.20 kcal/mol, respectively. These calculated free energy differences are in excellent agreement with the corresponding reported experimental values. Surprisingly, a significant contribution to the increased stability of the Hfl-containing fluorinated protein comes from van der Waals interactions and, in the case of Pff, electrostatic interactions. The simulated quantitative agreement with experimental values validates the free energy simulation method to elucidate the effect of fluorination on protein stability. The implications of the simulation results for our understanding of the effect of fluorination on protein stability are discussed.

Application of a Highly Selective Cathepsin S Two-step Activity-Based Probe in Multicolor Bio-Orthogonal Correlative Light-Electron Microscopy.

Cathepsin S is a lysosomal cysteine protease highly expressed in immune cells such as dendritic cells, B cells and macrophages. Its functions include extracellular matrix breakdown and cleavage of cell adhesion molecules to facilitate immune cell motility, as well as cleavage of the invariant chain during maturation of major histocompatibility complex II. The identification of these diverse specific functions has brought the challenge of delineating cathepsin S activity with great spatial precision, relative to related enzymes and substrates. Here, the development of a potent and highly selective two-step activity-based probe for cathepsin S and the application in multicolor bio-orthogonal correlative light-electron microscopy is presented. LHVS, which has been reported as a selective inhibitor of cathepsin S with nanomolar potency, formed the basis for our probe design. However, in competitive activity-based protein profiling experiments LHVS showed significant cross-reactivity toward Cat L. Introduction of an azide group in the P2 position expanded the selectivity window for cathepsin S, but rendered the probe undetectable, as demonstrated in bio-orthogonal competitive activity-based protein profiling. Incorporation of an additional azide handle for click chemistry on the solvent-exposed P1 position allowed for selective labeling of cathepsin S. This highlights the influence of click handle positioning on probe efficacy. This probe was utilized in multicolor bio-orthogonal confocal and correlative light-electron microscopy to investigate the localization of cathepsin S activity at an ultrastructural level in bone marrow-derived dendritic cells. The tools developed in this study will aid the characterization of the variety of functions of cathepsin S throughout biology.

Red, Orange, Green: Light- and Temperature-Dependent Color Tuning in a Cyanobacteriochrome.

Cyanobacteriochromes (CBCRs) are photoreceptor proteins that photoconvert between two parent states and thereby regulate various biological processes. An intriguing property is their variable ultraviolet-visible (UV-vis) absorption that covers the entire spectral range from the far-red to the near-UV region and thus makes CBCRs promising candidates for optogenetic applications. Here, we have studied Slr1393, a CBCR that photoswitches between red- and green-absorbing states (Pr and Pg, respectively). Using UV-vis absorption, fluorescence, and resonance Raman (RR) spectroscopy, a further orange-absorbing state O600 that is in thermal equilibrium with Pr was identified. The different absorption properties of the three states were attributed to the different lengths of the conjugated π-electron system of the phycocyanobilin chromophore. In agreement with available crystal structures and supported by quantum mechanics/molecular mechanics (QM/MM) calculations, the most extended conjugation holds for Pr whereas it is substantially reduced in Pg. Here, the two outer pyrrole rings D and A are twisted out of the plane defined by inner pyrrole rings B and C. For the O600 state, the comparison of the experimental RR spectra with QM/MM-calculated spectra indicates a partially distorted ZZZssa geometry in which ring A is twisted while ring D and the adjacent methine bridge display essentially the same geometry as Pr. The quantitative analysis of temperature-dependent spectra yields an enthalpy barrier of ∼30 kJ/mol for the transition from Pr to O600. This reaction is associated with the movement of a conserved tryptophan residue from the chromophore binding pocket to a solvent-exposed position.

Structural Features on the Substrate-Binding Surface of Fungal Lytic Polysaccharide Monooxygenases Determine Their Oxidative Regioselectivity.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidatively cleave many of nature's most recalcitrant polysaccharides by acting on the C1- and/or C4-carbon of the glycosidic bond. Here, the results of an extensive mutagenesis study on three LPMO representatives, Phanerochaete chrysosporium LPMO9D (C1-oxidizer), Neurospora crassa LPMO9C (C4), and Hypocrea jecorina LPMO9A (C1/C4), are reported. Using a previously published indicator diagram, the authors demonstrate that several structural determinants of LPMOs play an important role in their oxidative regioselectivity. N-glycan removal and alterations of the aromatic residues on the substrate-binding surface are shown to alter C1/C4-oxidation ratios. Removing the carbohydrate binding module (CBM) is found not to alter the regioselectivity of HjLPMO9A, although the effect of mutational changes is shown to increase in a CBM-free context. The accessibility to the solvent-exposed axial position of the copper-site reveales not to be a major regioselectivity indicator, at least not in PcLPMO9D. Interestingly, a HjLPMO9A variant lacking two surface exposed aromatic residues combines decreased binding capacity with a 22% increase in synergetic efficiency. Similarly to recent LPMO10 findings, our results suggest a complex matrix of surface-interactions that enables LPMO9s not only to bind their substrate, but also to accurately direct their oxidative force.

Paying the Price of Desolvation in Solvent-Exposed Protein Pockets: Impact of Distal Solubilizing Groups on Affinity and Binding Thermodynamics in a Series of Thermolysin Inhibitors.

In lead optimization, open, solvent-exposed protein pockets are often disregarded as prospective binding sites. Because of bulk-solvent proximity, researchers are instead enticed to attach charged polar groups at inhibitor scaffolds to improve solubility and pharmacokinetic properties. It is rarely considered that solvent effects from water reorganization in the first hydration shell of protein-ligand complexes can have a significant impact on binding. We investigate the thermodynamic fingerprint of thermolysin inhibitors featuring terminal charged ammonium groups that are gradually pulled from a distal, solvent-exposed position into the flat, bowl-shaped S2' pocket. Even for the most remote attachment, costs for partial desolvation of the polar group next to the protein-solvent interface are difficult to compensate by interactions with the protein or surrounding water molecules. Through direct comparison with hydrophobic analogues, a significant 180-fold affinity loss was recorded, which questions popular strategies to attach polar ligand-solubilizing groups at the exposed terminus of substituents accommodated in flat open pockets.

Natural and non-natural amino-acid side-chain substitutions: affinity and diffraction studies of meditope-Fab complexes.

Herein, multiple crystal structures of meditope peptide derivatives incorporating natural and unnatural amino acids bound to the cetuximab Fab domain are presented. The affinity of each derivative was determined by surface plasmon resonance and correlated to the atomic structure. Overall, it was observed that the hydrophobic residues in the meditope peptide, Phe3, Leu5 and Leu10, could accommodate a number of moderate substitutions, but these invariably reduced the overall affinity and half-life of the interaction. In one case, the substitution of Phe3 by histidine led to a change in the rotamer conformation, in which the imidazole ring flipped to a solvent-exposed position. Based on this observation, Phe3 was substituted by diphenylalanine and it was found that the phenyl rings in this variant mimic the superposition of the Phe3 and His3 structures, producing a moderate increase, of 1.4-fold, in the half-life of the complex. In addition, it was observed that substitution of Leu5 by tyrosine and glutamate strongly reduced the affinity, whereas the substitution of Leu5 by diphenylalanine moderately reduced the half-life (by approximately fivefold). Finally, it was observed that substitution of Arg8 and Arg9 by citrulline dramatically reduced the overall affinity, presumably owing to lost electrostatic interactions. Taken together, these studies provide insight into the meditope-cetuximab interaction at the atomic level.

Bioorthogonal Modification of the Major Sheath Protein of Bacteriophage M13: Extending the Versatility of Bionanomaterial Scaffolds.

With a mass of ∼1.6 × 107 Daltons and composed of approximately 2700 proteins, bacteriophage M13 has been employed as a molecular scaffold in bionanomaterials fabrication. In order to extend the versatility of M13 in this area, residue-specific unnatural amino acid incorporation was employed to successfully display azide functionalities on specific solvent-exposed positions of the pVIII major sheath protein of this bacteriophage. Employing a combination of engineered mutants of the gene coding for the pVIII protein, the methionine (Met) analog, l-azidohomoalanine (Aha), and a suitable Escherichia coli Met auxotroph for phage production, conditions were developed to produce M13 bacteriophage labeled with over 350 active azides (estimated by fluorescent dye labeling utilizing a strain-promoted azide-alkyne cycloaddition) and capable of azide-selective attachment to 5 nm gold nanoparticles as visualized by transmission electron microscopy. The capability of this system to undergo dual labeling utilizing both chemical acylation and bioorthogonal cycloaddition reactions was also verified. The above stratagem should prove particularly advantageous in the preparation of assemblies of larger and more complex molecular architectures based on the M13 building block.

Effects of Single α-to-β Residue Replacements on Structure and Stability in a Small Protein: Insights from Quasiracemic Crystallization.

Synthetic peptides that contain backbone modifications but nevertheless adopt folded structures similar to those of natural polypeptides are of fundamental interest and may provide a basis for biomedical applications. Such molecules can, for example, mimic the ability of natural prototypes to bind to specific target macromolecules but resist degradation by proteases. We have previously shown that oligomers containing mixtures of α- and β-amino acid residues ("α/β-peptides") can mimic the α-helix secondary structure, and that properly designed α/β-peptides can bind to proteins that evolved to bind to α-helical partners. Here we report fundamental studies that support the long-range goal of extending the α/β approach to tertiary structures. We have evaluated the impact of single α → β modifications on the structure and stability of the small and well-studied villin headpiece subdomain (VHP). The native state of this 35-residue polypeptide contains several α-helical segments packed around a small hydrophobic core. We examined α → β substitution at four solvent-exposed positions, Asn19, Trp23, Gln26 and Lys30. In each case, both the β(3) homologue of the natural α residue and a cyclic β residue were evaluated. All α → β(3) substitutions caused significant destabilization of the tertiary structure as measured by variable-temperature circular dichroism, although at some of these positions, replacing the β(3) residue with a cyclic β residue led to improved stability. Atomic-resolution structures of four VHP analogues were obtained via quasiracemic crystallization. These findings contribute to a fundamental α/β-peptide knowledge-base by confirming that β(3)-amino acid residues can serve as effective structural mimics of homologous α-amino acid residues within a natural tertiary fold, which should support rational design of functional α/β analogues of natural poly-α-peptides.

Site-specific labeling of proteins for electron microscopy.

Electron microscopy is commonly employed to determine the subunit organization of large macromolecular assemblies. However, the field lacks a robust molecular labeling methodology for unambiguous identification of constituent subunits. We present a strategy that exploits the unique properties of an unnatural amino acid in order to enable site-specific attachment of a single, readily identifiable protein label at any solvent-exposed position on the macromolecular surface. Using this method, we show clear labeling of a subunit within the 26S proteasome lid subcomplex that has not been amenable to labeling by traditional approaches.

Aromatic cluster mutations produce focal modulations of β-sheet structure.

Site-directed mutagenesis is a powerful tool for altering the structure and function of proteins in a focused manner. Here, we examined how a model β-sheet protein could be tuned by mutation of numerous surface-exposed residues to aromatic amino acids. We designed these aromatic side chain "clusters" at highly solvent-exposed positions in the flat, single-layer β-sheet of Borrelia outer surface protein A (OspA). This unusual β-sheet scaffold allows us to interrogate the effects of these mutations in the context of well-defined structure but in the absence of the strong scaffolding effects of globular protein architecture. We anticipated that the introduction of a cluster of aromatic amino acid residues on the β-sheet surface would result in large conformational changes and/or stabilization and thereby provide new means of controlling the properties of β-sheets. Surprisingly, X-ray crystal structures revealed that the introduction of aromatic clusters produced only subtle conformational changes in the OspA β-sheet. Additionally, despite burying a large degree of hydrophobic surface area, the aromatic cluster mutants were slightly less stable than the wild-type scaffold. These results thereby demonstrate that the introduction of aromatic cluster mutations can serve as a means for subtly modulating β-sheet conformation in protein design.

Cysi–Lysi+3–Lysi+4 Triad: A General Approach for PEG-Based Stabilization of α-Helical Proteins

PEGylation is an important strategy for enhancing the pharmacokinetic properties of protein drugs. Modern chemoselective reactions now enable specific placement of a single PEG at any site on a protein surface. However, few rational structure-based guidelines exist for selecting optimal PEGylation sites. Here, we explore the impact of PEGylation on the conformational stability of α-helices using an α-helical coiled coil as a model system. We find that maleimide-based PEGylation of a solvent-exposed i position Cys can stabilize coiled-coil quaternary structure when Lys residues occupy both the i + 3 and i + 4 positions, due to favorable interactions between the PEG-maleimide and the Lys residues. Applying this Cys(i)-Lys(i+3)-Lys(i+4) triad to a solvent-exposed position within the C-terminal helix of the villin headpiece domain leads to similar PEG-based increases in conformational stability, highlighting the possibility of using the Cys(i)-Lys(i+3)-Lys(i+4) triad as a general strategy for PEG-based stabilization of helical proteins.

A systematic survey of an intragenic epistatic landscape.

Mutations are the source of evolutionary variation. The interactions of multiple mutations can have important effects on fitness and evolutionary trajectories. We have recently described the distribution of fitness effects of all single mutations for a nine-amino-acid region of yeast Hsp90 (Hsp82) implicated in substrate binding. Here, we report and discuss the distribution of intragenic epistatic effects within this region in seven Hsp90 point mutant backgrounds of neutral to slightly deleterious effect, resulting in an analysis of more than 1,000 double mutants. We find negative epistasis between substitutions to be common, and positive epistasis to be rare--resulting in a pattern that indicates a drastic change in the distribution of fitness effects one step away from the wild type. This can be well explained by a concave relationship between phenotype and genotype (i.e., a concave shape of the local fitness landscape), suggesting mutational robustness intrinsic to the local sequence space. Structural analyses indicate that, in this region, epistatic effects are most pronounced when a solvent-inaccessible position is involved in the interaction. In contrast, all 18 observations of positive epistasis involved at least one mutation at a solvent-exposed position. By combining the analysis of evolutionary and biophysical properties of an epistatic landscape, these results contribute to a more detailed understanding of the complexity of protein evolution.

Engineered solubility tag for solution NMR of proteins

The low solubility of many proteins hinders large scale expression and purification as well as biophysical measurements. Here, we devised a general strategy to solubilize a protein by conjugating it at a solvent-exposed position to a 6 kDa protein that was re-engineered to be highly soluble. We applied this method to the CARD domain of Apoptosis-associated speck-like protein containing a CARD (ASC), which represents one member of a class of proteins that are notoriously prone to aggregation. Attachment of the tag to a cysteine residue, introduced by site-directed mutagenesis at its self-association interface, improved the solubility of the ASC CARD over 50-fold under physiological conditions. Although it is not possible to use nuclear magnetic resonance (NMR) to obtain a high quality 2D correlation spectrum of the wild type domain under physiological conditions, we demonstrate that NMR relaxation parameters of the solubilized variant are sufficiently improved to facilitate virtually any demanding measurement. The method shown here represents a straightforward approach for dramatically increasing protein solubility, enabled by ease of labeling as well as flexibility in tag placement with minimal perturbation to the target.

Oxime Side‐Chain Cross‐Links in an α‐Helical Coiled‐Coil Protein: Structure, Thermodynamics, and Folding‐Templated Synthesis of Bicyclic Species

Covalent side-chain cross-links are a versatile method to control peptide folding, particularly when α-helical secondary structure is the target. Here, we examine the application of oxime bridges, formed by the chemoselective reaction between aminooxy and aldehyde side chains, for the stabilization of a helical peptide involved in a protein-protein complex. A series of sequence variants of the dimeric coiled coil GCN4-p1 bearing oxime bridges at solvent-exposed positions were prepared and biophysically characterized. Triggered unmasking of a side-chain aldehyde in situ and subsequent cyclization proceed rapidly and cleanly at pH 7 in the folded protein complex. Comparison of folding thermodynamics among a series of different oxime bridges show that the cross links are consistently stabilizing to the coiled coil, with the extent of stabilization sensitive to the exact size and structure of the macrocycle. X-ray crystallographic analysis of a coiled coil with the best cross link in place and a second structure of its linear precursor show how the bridge is accommodated into an α-helix. Preparation of a bicyclic oligomer by simultaneous formation of two linkages in situ demonstrates the potential use of triggered oxime formation to both trap and stabilize a particular peptide folded conformation in the bound state.

A polymetamorphic protein

Arc repressor is a homodimeric protein with a ribbon-helix-helix fold. A single polar-to-hydrophobic substitution (N11L) at a solvent-exposed position leads to population of an alternate dimeric fold in which 3₁₀ helices replace a β-sheet. Here we find that the variant Q9V/N11L/R13V (S-VLV), with two additional polar-to-hydrophobic surface mutations in the same β-sheet, forms a highly stable, reversibly folded octamer with approximately half the α-helical content of wild-type Arc. At low protein concentration and low ionic strength, S-VLV also populates both dimeric topologies previously observed for N11L, as judged by NMR chemical shift comparisons. Thus, accumulation of simple hydrophobic mutations in Arc progressively reduces fold specificity, leading first to a sequence with two folds and then to a manifold bridge sequence with at least three different topologies. Residues 9-14 of S-VLV form a highly hydrophobic stretch that is predicted to be amyloidogenic, but we do not observe aggregates of higher order than octamer. Increases in sequence hydrophobicity can promote amyloid aggregation but also exert broader and more complex effects on fold specificity. Altered native folds, changes in fold coupled to oligomerization, toxic pre-amyloid oligomers, and amyloid fibrils may represent a near continuum of accessible alternatives in protein structure space.

Visualizing transient protein-folding intermediates by tryptophan-scanning mutagenesis

To understand how proteins fold, assemble and function, it is necessary to characterize the structure and dynamics of each state they adopt during their lifetime. Experimental characterization of the transient states of proteins remains a major challenge because high-resolution structural techniques, including NMR and X-ray crystallography, cannot be directly applied to study short-lived protein states. To circumvent this limitation, we show that transient states during protein folding can be characterized by measuring the fluorescence of tryptophan residues, introduced at many solvent-exposed positions to determine whether each position is native-like, denatured-like or non-native-like in the intermediate state. We use this approach to characterize a late-folding-intermediate state of the small globular mammalian protein ubiquitin, and we show the presence of productive non-native interactions that suggest a 'flycatcher' mechanism of concerted binding and folding.

Insights into the Mechanism of Bovine CD38/NAD+Glycohydrolase from the X-Ray Structures of Its Michaelis Complex and Covalently-Trapped Intermediates

Bovine CD38/NAD+glycohydrolase (bCD38) catalyses the hydrolysis of NAD+ into nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose (cADPR). We solved the crystal structures of the mono N-glycosylated forms of the ecto-domain of bCD38 or the catalytic residue mutant Glu218Gln in their apo state or bound to aFNAD or rFNAD, two 2′-fluorinated analogs of NAD+. Both compounds behave as mechanism-based inhibitors, allowing the trapping of a reaction intermediate covalently linked to Glu218. Compared to the non-covalent (Michaelis) complex, the ligands adopt a more folded conformation in the covalent complexes. Altogether these crystallographic snapshots along the reaction pathway reveal the drastic conformational rearrangements undergone by the ligand during catalysis with the repositioning of its adenine ring from a solvent-exposed position stacked against Trp168 to a more buried position stacked against Trp181. This adenine flipping between conserved tryptophans is a prerequisite for the proper positioning of the N1 of the adenine ring to perform the nucleophilic attack on the C1′ of the ribofuranoside ring ultimately yielding cADPR. In all structures, however, the adenine ring adopts the most thermodynamically favorable anti conformation, explaining why cyclization, which requires a syn conformation, remains a rare alternate event in the reactions catalyzed by bCD38 (cADPR represents only 1% of the reaction products). In the Michaelis complex, the substrate is bound in a constrained conformation; the enzyme uses this ground-state destabilization, in addition to a hydrophobic environment and desolvation of the nicotinamide-ribosyl bond, to destabilize the scissile bond leading to the formation of a ribooxocarbenium ion intermediate. The Glu218 side chain stabilizes this reaction intermediate and plays another important role during catalysis by polarizing the 2′-OH of the substrate NAD+. Based on our structural analysis and data on active site mutants, we propose a detailed analysis of the catalytic mechanism.

Methionine Scanning as an NMR Tool for Detecting and Analyzing Biomolecular Interaction Surfaces

Methyl NMR spectroscopy is a powerful tool for studying protein structure, dynamics, and interactions. Yet difficulties with resonance assignment and the low abundance of methyl groups can preclude detailed NMR studies, particularly the determination of continuous interaction surfaces. Here we present a straightforward strategy that overcomes these problems. We systematically substituted solvent-exposed residues with reporter methionines in the expected binding site and performed chemical shift perturbation (CSP) experiments using methyl-TROSY spectra. We demonstrate the utility of this approach for the interaction between the HECT domain of the Rsp5p ubiquitin ligase and its cognate E2, Ubc4. Using these mutants, we could instantaneously assign all newly arising reporter methyl signals, determine the Ubc4 interaction surface on a per-residue basis, and investigate the importance of each individual mutation for ligand binding. Our data show that methionine scanning significantly extends the applicability, information content, and spatial resolution of methyl CSP experiments.

Fundamental Characteristics of the Immunoglobulin VH Repertoire of Chickens in Comparison with Those of Humans, Mice, and Camelids

Examination of 1269 unique naive chicken V(H) sequences showed that the majority of positions in the framework (FW) regions were maintained as germline, with high mutation rates observed in the CDRs. Many FW mutations could be clearly related to the modulation of CDR structure or the V(H)-V(L) interface. CDRs 1 and 2 of the V(H) exhibited frequent mutation in solvent-exposed positions, but conservation of common structural residues also found in human CDRs at the same positions. In comparison with humans and mice, the chicken CDR3 repertoire was skewed toward longer sequences, was dominated by small amino acids (G/S/A/C/T), and had higher cysteine (chicken, 9.4%; human, 1.6%; and mouse, 0.25%) but lower tyrosine content (chicken, 9.2%; human, 16.8%; and mouse 26.4%). A strong correlation (R(2) = 0.97) was observed between increasing CDR3 length and higher cysteine content. This suggests that noncanonical disulfides are strongly favored in chickens, potentially increasing CDR stability and complexity in the topology of the combining site. The probable formation of disulfide bonds between CDR3 and CDR1, FW2, or CDR2 was also observed, as described in camelids. All features of the naive repertoire were fully replicated in the target-selected, phage-displayed repertoire. The isolation of a chicken Fab with four noncanonical cysteines in the V(H) that exhibits 64 nM (K(D)) binding affinity for its target proved these constituents to be part of the humoral response, not artifacts. This study supports the hypothesis that disulfide bond-constrained CDR3s are a structural diversification strategy in the restricted germline v-gene repertoire of chickens.