Biophysical Properties of Camelid VHH Domains Compared to Those of Human VH3 Domains

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Abstract
Camelidae possess an unusual form of antibodies lacking the light chains. The variable domain of these heavy chain antibodies (VHH) is not paired, while the VH domain of all other antibodies forms a heterodimer with the variable domain of the light chain (VL), held together by a hydrophobic interface. Here, we analyzed the biophysical properties of four camelid VHH fragments (H14, AMD9, RN05, and CA05) and two human consensus VH3 domains with different CDR3 loops to gain insight into factors determining stability and aggregation of immunoglobulin domains. We show by denaturant-induced unfolding equilibria that the free energies of unfolding of VHH fragments are characterized by ΔGN-U values between 21.1 and 35.0 kJ/mol and thus lie in the upper range of values for VH fragments from murine and human antibodies. Nevertheless, the VHH fragments studied here did not reach the high values between 39.7 and 52.7 kJ/mol of the human consensus VH3 domains with which they share the highest degree of sequence similarity. Temperature-induced unfolding of the VHH fragments that were studied proved to be reversible, and the binding affinity after cooling was fully retained. The melting temperatures were determined to be between 60.1 and 66.7 °C. In contrast, the studied VH3 domains aggregated during temperature-induced denaturation at 63−65 °C. In summary, the camelid VHH fragments are characterized by a favorable but not unusually high stability. Their hallmark is the ability to reversibly melt without aggregation, probably mediated by the surface mutations characterizing the VHH domains, which allow them to regain binding activity after heat renaturation.