http://experimentationlab.berkeley.edu/sites/default/files/AFMImages/butt_AFM.pdf

Force measurements with the atomic force microscope: Technique, interpretation and applications

Antibodies, the most popular class of molecules providing molecular recognition needs for a wide range of applications, have been around for more than three decades. As a result, antibodies have made substantial contributions toward the advancement of diagnostic assays and have become indispensable in most diagnostic tests that are used routinely in clinics today. The development of the systematic evolution of ligands by exponential enrichment (SELEX) process, however, made possible the isolation of oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. These oligonucleotide sequences, referred to as "aptamers", are beginning to emerge as a class of molecules that rival antibodies in both therapeutic and diagnostic applications. Aptamers are different from antibodies, yet they mimic properties of antibodies in a variety of diagnostic formats. The demand for diagnostic assays to assist in the management of existing and emerging diseases is increasing, and aptamers could potentially fulfill molecular recognition needs in those assays. Compared with the bellwether antibody technology, aptamer research is still in its infancy, but it is progressing at a fast pace. The potential of aptamers may be realized in the near future in the form of aptamer-based diagnostic products in the market. In such products, aptamers may play a key role either in conjunction with, or in place of, antibodies. It is also likely that existing diagnostic formats may change according to the need to better harness the unique properties of aptamers.

Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics

Molecular recognition is central to all biological processes. For the past 50 years, Koshland's 'induced fit' hypothesis has been the textbook explanation for molecular recognition events. However, recent experimental evidence supports an alternative mechanism. 'Conformational selection' postulates that all protein conformations pre-exist, and the ligand selects the most favored conformation. Following binding the ensemble undergoes a population shift, redistributing the conformational states. Both conformational selection and induced fit appear to play roles. Following binding by a primary conformational selection event, optimization of side chain and backbone interactions is likely to proceed by an induced fit mechanism. Conformational selection has been observed for protein-ligand, protein-protein, protein-DNA, protein-RNA and RNA-ligand interactions. These data support a new molecular recognition paradigm for processes as diverse as signaling, catalysis, gene regulation and protein aggregation in disease, which has the potential to significantly impact our views and strategies in drug design, biomolecular engineering and molecular evolution.

/pdf/the-role-of-dynamic-conformational-ensembles-in-biomolecular-33rwd16x5b.pdf

The role of dynamic conformational ensembles in biomolecular recognition

We have developed a protein-synthesizing system reconstituted from recombinant tagged protein factors purified to homogeneity. The system was able to produce protein at a rate of about 160 μg/ml/h in a batch mode without the need for any supplementary apparatus. The protein products were easily purified within 1 h using affinity chromatography to remove the tagged protein factors. Moreover, omission of a release factor allowed efficient incorporation of an unnatural amino acid using suppressor transfer RNA (tRNA).

/pdf/cell-free-translation-reconstituted-with-purified-components-3yqtgmhg62.pdf

Cell-free translation reconstituted with purified components

Sera of camelids contain both conventional heterotetrameric antibodies and unique functional heavy (H)-chain antibodies (HCAbs). The H chain of these homodimeric antibodies consists of one antigen-binding domain, the VHH, and two constant domains. HCAbs fail to incorporate light (L) chains owing to the deletion of the first constant domain and a reshaped surface at the VHH side, which normally associates with L chains in conventional antibodies. The genetic elements composing HCAbs have been identified, but the in vivo generation of these antibodies from their dedicated genes into antigen-specific and affinity-matured bona fide antibodies remains largely underinvestigated. However, the facile identification of antigen-specific VHHs and their beneficial biochemical and economic properties (size, affinity, specificity, stability, production cost) supported by multiple crystal structures have encouraged antibody engineering of these single-domain antibodies for use as a research tool and in biotechnology and medicine.

Nanobodies: Natural Single-Domain Antibodies

Ribosome display was applied for affinity selection of antibody single-chain fragments (scFv) from a diverse library generated from mice immunized with a variant peptide of the transcription factor GCN4 dimerization domain. After three rounds of ribosome display, positive scFvs were isolated and characterized. Several different scFvs were selected, but those in the largest group were closely related to each other and differed in 0 to 5 amino acid residues with respect to their consensus sequence, the likely common progenitor. The best scFv had a dissociation constant of (4 ± 1) × 10−11 M, measured in solution. One amino acid residue in complementarity determining region L1 was found to be responsible for a 65-fold higher affinity than the likely progenitor. It appears that this high-affinity scFv was selected from the mutations occurring during ribosome display in vitro, and that this constitutes an affinity maturation inherent in this method. The in vitro-selected scFvs could be functionally expressed in the Escherichia coli periplasm with good yields or prepared by in vitro refolding. Thus, ribosome display can be a powerful methodology for in vitro library screening and simultaneous sequence evolution.

https://www.pnas.org/content/pnas/95/24/14130.full.pdf

Ribosome Display Efficiently Selects and Evolves High-Affinity Antibodies in vitro from Immune Libraries

Point mutants of three unrelated antifluorescein antibodies were constructed to obtain nine different single-chain Fv fragments, whose on-rates, off-rates, and equilibrium binding affinities were determined in solution. Additionally, activation energies for unbinding were estimated from the temperature dependence of the off-rate in solution. Loading rate-dependent unbinding forces were determined for single molecules by atomic force microscopy, which extrapolated at zero force to a value close to the off-rate measured in solution, without any indication for multiple transition states. The measured unbinding forces of all nine mutants correlated well with the off-rate in solution, but not with the temperature dependence of the reaction, indicating that the same transition state must be crossed in spontaneous and forced unbinding and that the unbinding path under load cannot be too different from the one at zero force. The distance of the transition state from the ground state along the unbinding pathway is directly proportional to the barrier height, regardless of the details of the binding site, which most likely reflects the elasticity of the protein in the unbinding process. Atomic force microscopy thus can be a valuable tool for the characterization of solution properties of protein-ligand systems at the single molecule level, predicting relative off-rates, potentially of great value for combinatorial chemistry and biology.

/pdf/unbinding-forces-of-single-antibody-antigen-complexes-36pnmccyyu.pdf

Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates

Ribosome display: an in vitro method for selection and evolution of antibodies from libraries.

We describe a rapid and general technology working entirely in vitro to evolve either the affinity or the stability of ligand-binding proteins, depending on the chosen selection pressure Tailored in vitro selection strategies based on ribosome display were combined with in vitro diversification by DNA shuffling to evolve either the off-rate or thermodynamic stability of single-chain Fv antibody fragments (scFvs) To demonstrate the potential of this method, we chose to optimize two proteins already possessing favorable properties A scFv with an initial affinity of 11 nM (koff at 4°C of 10−4 s−1) was improved 30-fold by the use of off-rate selections over a period of several days As a second example, a generic selection strategy for improved stability exploited the property of ribosome display that the conditions can be altered under which the folding of the displayed protein occurs We used decreasing redox potentials in the selection step to select for molecules stable in the absence of disulfide bonds They could be functionally expressed in the reducing cytoplasm, and, when allowed to form disulfides again, their stability had increased to 54 kJ/mol from an initial value of 24 kJ/mol Sequencing revealed that the evolved mutant proteins had used different strategies of residue changes to adapt to the selection pressure Therefore, by a combination of randomization and appropriate selection strategies, an in vitro evolution of protein properties in a predictable direction is possible

https://www.pnas.org/content/pnas/98/1/75.full.pdf

Lutz Jermutus

Papers

Ribosome Display Efficiently Selects and Evolves High-Affinity Antibodies in vitro from Immune Libraries

Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates

Ribosome display: an in vitro method for selection and evolution of antibodies from libraries.

Tailoring in vitro evolution for protein affinity or stability

Recent advances in producing and selecting functional proteins by using cell-free translation