Sheldon Park

Streptavidin–biotin technology: improvements and innovations in chemical and biological applications

Streptavidin and its homologs (together referred to as streptavidin) are widely used in molecular science owing to their highly selective and stable interaction with biotin. Other factors also contribute to the popularity of the streptavidin–biotin system, including the stability of the protein and various chemical and enzymatic biotinylation methods available for use with different experimental designs. The technology has enjoyed a renaissance of a sort in recent years, as new streptavidin variants are engineered to complement native proteins and novel methods of introducing selective biotinylation are developed for in vitro and in vivo applications. There have been notable developments in the areas of catalysis, cell biology, and proteomics in addition to continued applications in the more established areas of detection, labeling and drug delivery. This review summarizes recent advances in streptavidin engineering and new applications based on the streptavidin–biotin interaction.

Statistical and molecular dynamics studies of buried waters in globular proteins

Buried solvent molecules are common in the core of globular proteins and contribute to structural stability. Folding necessitates the burial of polar backbone atoms in the protein core, whose hydrogen‐bonding capacities should be satisfied on average. Whereas the residues in α‐helices and β‐sheets form systematic main‐chain hydrogen bonds, the residues in turns, coils and loops often contain polar atoms that fail to form intramolecular hydrogen bonds. The statistical analysis of 842 high resolution protein structures shows that well‐resolved, internal water molecules preferentially reside near residues without α‐helical and β‐sheet secondary structures. These buried waters most often form primary hydrogen bonds to main‐chain atoms not involved in intramolecular hydrogen bonds, providing strong evidence that hydrating main‐chain atoms is a key structural role of buried water molecules. Additionally, the average B‐factor of protein atoms hydrogen‐bonded to waters is smaller than that of protein atoms forming intramolecular hydrogen bonds, and the average B‐factor of water molecules involved in primary hydrogen bonds with main‐chain atoms is smaller than the average B‐factor of water molecules involved in secondary hydrogen bonds to protein atoms that form concurrent intramolecular hydrogen bonds. To study the structural coupling between internal waters and buried polar atoms in detail we simulated the dynamics of wild‐type FKBP12, in which a buried water, Wat137, forms one side‐chain and multiple main‐chain hydrogen bonds. We mutated E60, whose side‐chain hydrogen bonds with Wat137, to Q, N, S or A, to modulate the multiplicity and geometry of hydrogen bonds to the water. Mutating E60 to a residue that is unable to form a hydrogen bond with Wat137 results in reorientation of the water molecule and leads to a structural readjustment of residues that are both near and distant to the water. We predict that the E60A mutation will result in a significantly reduced affinity of FKBP12 for its ligand FK506. The propensity of internal waters to hydrogen bond to buried polar atoms suggests that ordered water molecules may constitute fundamental structural components of proteins, particularly in regions where α‐helical or β‐sheet secondary structure is not present. Proteins 2005.

Structural coupling between FKBP12 and buried water.

Globular proteins often contain structurally well‐resolved internal water molecules. Previously, we reported results from a molecular dynamics study that suggested that buried water (Wat3) may play a role in modulating the structure of the FK506 binding protein‐12 (FKBP12) (Park and Saven, Proteins 2005; 60:450–463). In particular, simulations suggested that disrupting a hydrogen bond to Wat3 by mutating E60 to either A or Q would cause a structural perturbation involving the distant W59 side chain, which rotates to a new conformation in response to the mutation. This effectively remodels the ligand‐binding pocket, as the side chain in the new conformation is likely to clash with bound FK506. To test whether the protein structure is in effect modulated by the binding of a buried water in the distance, we determined high‐resolution (0.92–1.29 Å) structures of wild‐type FKBP12 and its two mutants (E60A, E60Q) by X‐ray crystallography. The structures of mutant FKBP12 show that the ligand‐binding pocket is indeed remodeled as predicted by the substitution at position 60, even though the water molecule does not directly interact with any of the amino acids of the binding pocket. Thus, these structures support the view that buried water molecules constitute an integral, noncovalent component of the protein structure. Additionally, this study provides an example in which predictions from molecular dynamics simulations are experimentally validated with atomic precision, thus showing that the structural features of protein–water interactions can be reliably modeled at a molecular level. Proteins 2009.

Stable, high‐affinity streptavidin monomer for protein labeling and monovalent biotin detection

The coupling between the quaternary structure, stability and function of streptavidin makes it difficult to engineer a stable, high affinity monomer for biotechnology applications. For example, the binding pocket of streptavidin tetramer is comprised of residues from multiple subunits, which cannot be replicated in a single domain protein. However, rhizavidin from Rhizobium etli was recently shown to bind biotin with high affinity as a dimer without the hydrophobic tryptophan lid donated by an adjacent subunit. In particular, the binding site of rhizavidin uses residues from a single subunit to interact with bound biotin. We therefore postulated that replacing the binding site residues of streptavidin monomer with corresponding rhizavidin residues would lead to the design of a high affinity monomer useful for biotechnology applications. Here, we report the construction and characterization of a structural monomer, mSA, which combines the streptavidin and rhizavidin sequences to achieve optimized biophysical properties. First, the biotin affinity of mSA (Kd = 2.8 nM) is the highest among nontetrameric streptavidin, allowing sensitive monovalent detection of biotinylated ligands. The monomer also has significantly higher stability (Tm = 59.8°C) and solubility than all other previously engineered monomers to ensure the molecule remains folded and functional during its application. Using fluorescence correlation spectroscopy, we show that mSA binds biotinylated targets as a monomer. We also show that the molecule can be used as a genetic tag to introduce biotin binding capability to a heterologous protein. For example, recombinantly fusing the monomer to a cell surface receptor allows direct labeling and imaging of transfected cells using biotinylated fluorophores. A stable and functional streptavidin monomer, such as mSA, should be a useful reagent for designing novel detection systems based on monovalent biotin interaction. Biotechnol. Bioeng. 2013; 110: 57–67.

Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin

The advent of super-resolution imaging (SRI) has created a need for optimized labelling strategies. We present a new method relying on fluorophore-conjugated monomeric streptavidin (mSA) to label membrane proteins carrying a short, enzymatically biotinylated tag, compatible with SRI techniques including uPAINT, STED and dSTORM. We demonstrate efficient and specific labelling of target proteins in confined intercellular and organotypic tissues, with reduced steric hindrance and no crosslinking compared with multivalent probes. We use mSA to decipher the dynamics and nanoscale organization of the synaptic adhesion molecules neurexin-1β, neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2) in a dual-colour configuration with GFP nanobody, and show that these proteins are diffusionally trapped at synapses where they form apposed trans-synaptic adhesive structures. Furthermore, Nlg1 is dynamic, disperse and sensitive to synaptic stimulation, whereas LRRTM2 is organized in compact and stable nanodomains. Thus, mSA is a versatile tool to image membrane proteins at high resolution in complex live environments, providing novel information about the nano-organization of biological structures.

Engineered Streptavidin Monomer and Dimer with Improved Stability and Function

Although streptavidins high affinity for biotin has made it a widely used and studied binding protein and labeling tool, its tetrameric structure may interfere with some assays. A streptavidin mutant with a simpler quaternary structure would demonstrate a molecular-level understanding of its structural organization and lead to the development of a novel molecular reagent. However, modulating the tetrameric structure without disrupting biotin binding has been extremely difficult. In this study, we describe the design of a stable monomer that binds biotin both in vitro and in vivo. To this end, we constructed and characterized monomers containing rationally designed mutations. The mutations improved the stability of the monomer (increase in T(m) from 31 to 47 °C) as well as its affinity (increase in K(d) from 123 to 38 nM). We also used the stability-improved monomer to construct a dimer consisting of two streptavidin subunits that interact across the dimer-dimer interface, which we call the A/D dimer. The biotin binding pocket is conserved between the tetramer and the A/D dimer, and therefore, the dimer is expected to have a significantly higher affinity than the monomer. The affinity of the dimer (K(d) = 17 nM) is higher than that of the monomer but is still many orders of magnitude lower than that of the wild-type tetramer, which suggests there are other factors important for high-affinity biotin binding. We show that the engineered streptavidin monomer and dimer can selectively bind biotinylated targets in vivo by labeling the cells displaying biotinylated receptors. Therefore, the designed mutants may be useful in novel applications as well as in future studies in elucidating the role of oligomerization in streptavidin function.

Protein engineering and design

Phage Display Systems for Protein Engineering, A. Ernst and S. S. Sidhu Cell Surface Display Systems for Protein Engineering, S. J. Moore, M. J. Olsen, J. R. Cochran, and F. V. Cochran Cell-Free Display Systems for Protein Engineering, P. A. Barendt and C. A. Sarkar Library Construction for Protein Engineering, D. Lipovsek, M. Mena, S. M. Lippow, S. Basu, and B. M. Baynes Design and Engineering of Synthetic Binding Proteins Using Nonantibody Scaffolds, S. Koide Combinatorial Enzyme Engineering, P. C. Cirino and C. S. Frei Engineering of Therapeutic Proteins, F. Wen, S. B. Rubin-Pitel, and H. Zhao Protein Engineered Biomaterials, C. W. P. Foo and S. C. Heilshorn Protein Engineering Using Noncanonical Amino Acids, D. Yuksel , D. Pamuk, Y. Ivanova, and K. Kumar Computer Graphics, Homology Modeling, and Bioinformatics, D. F. Green Knowledge-Based Protein Design, M. A. Fisher, S. C. Patel, I. Cherny, and M. H. Hecht Molecular Force Fields, P. Koehl Rotamer Libraries for Molecular Modeling and Design of Proteins, H. Kono Search Algorithms, J. M. Shifman and M. Fromer Modulating Protein Structure, M. S. Hanes, T. M. Handel, and A. B. Chowdry Modulation of Intrinsic Properties by Computational Design, V. Nanda, F. Xu, and D. Hsieh Modulating Protein Interactions by Rational and Computational Design, J. S. Marvin and L. L. Looger Future Challenges of Computational Protein Design, E. J. Choi, G. Guntas, and B. Kuhlman

Progress in the development and application of computational methods for probabilistic protein design

Proteins exhibit a wide range of physical and chemical properties, including highly selective molecular recognition and catalysis, and are also key components in biological metabolic, catabolic, and signaling pathways. Given that proteins are well-structured and can be rapidly synthesized, they are excellent targets for engineering both molecular structure and biological function. Computational analysis of the protein design problem allows scientists to explore sequence space and systematically discover novel protein molecules. Nonetheless, the complexity of proteins, the subtlety of the determinants of folding, and the exponentially large number of possible sequences impede the search for peptide sequences compatible with a desired structure and function. Directed search algorithms, which identify directly a small number of sequences, have achieved some success in identifying sequences with desired structures and functions. Alternatively, one can adopt a probabilistic approach. Instead of a finite number of sequences, such calculations result in a probabilistic description of the sequence ensemble. In particular, by casting the formalism in the language of statistical mechanics, the site-specific amino acid probabilities of sequences compatible with a target structure may be readily estimated. These computed probabilities are well suited for both de novo protein design of particular sequences as well as combinatorial, library-based protein engineering. The computed site-specific amino acid profile may be converted to a nucleotide base distribution to allow assembly of a partially randomized gene library. The ability to synthesize readily such degenerate oligonucleotide sequences according to the prescribed distribution is key to constructing a biased peptide library genuinely reflective of the computational design. Herein we illustrate how a standard DNA synthesizer can be used with only a slight modification to the synthesis protocol to generate a pool of degenerate DNA sequences, which encodes a predetermined amino acid distribution with high fidelity.

Computational design of protein therapeutics

Computation is increasingly used to guide protein therapeutic designs. Some of the potential applications for computational, structure-based protein design include antibody affinity maturation, modulation of protein-protein interaction, stability improvement and minimization of protein aggregation. The versatility of a computational approach is that different biophysical properties can be analyzed on a common framework. Developing a coherent strategy to address various protein engineering objectives will promote synergy and exploration. Advances in computational structural analysis will thus have a transformative impact on how protein therapeutics are engineered in the future.:

Structure‐based engineering of streptavidin monomer with a reduced biotin dissociation rate

We recently reported the engineering of monomeric streptavidin, mSA, corresponding to one subunit of wild type (wt) streptavidin tetramer. The monomer was designed by homology modeling, in which the streptavidin and rhizavidin sequences were combined to engineer a high affinity binding pocket containing residues from a single subunit only. Although mSA is stable and binds biotin with nanomolar affinity, its fast off rate (koff) creates practical challenges during applications. We obtained a 1.9 Å crystal structure of mSA bound to biotin to understand their interaction in detail, and used the structure to introduce targeted mutations to improve its binding kinetics. To this end, we compared mSA to shwanavidin, which contains a hydrophobic lid containing F43 in the binding pocket and binds biotin tightly. However, the T48F mutation in mSA, which introduces a comparable hydrophobic lid, only resulted in a modest 20–40% improvement in the measured koff. On the other hand, introducing the S25H mutation near the bicyclic ring of bound biotin increased the dissociation half life (t½) from 11 to 83 min at 20°C. Molecular dynamics (MD) simulations suggest that H25 stabilizes the binding loop L3,4 by interacting with A47, and protects key intermolecular hydrogen bonds by limiting solvent entry into the binding pocket. Concurrent T48F or T48W mutation clashes with H25 and partially abrogates the beneficial effects of H25. Taken together, this study suggests that stabilization of the binding loop and solvation of the binding pocket are important determinants of the dissociation kinetics in mSA. Proteins 2013.