Folding of Flexible Protein Fragments and Design of Nanoparticle-Based Artificial Antibody Targeting Lysozyme

Abstract

It is generally believed that a protein’s sequence solely determines its native structure, but how the long- and short-range interactions jointly determine the native structure/conformation of the protein or every local fragment of the protein is still not fully understood. Since most protein fragments are unstructured on their own, direct observation of the folding of flexible protein fragments is very difficult. Interestingly, we show that it is possible to graft the complementary-determining regions (CDRs) of antibodies onto the surface of a gold nanoparticle (AuNP) to create AuNP-based artificial antibodies (denoted as Goldbodies), such as an antilysozyme Goldbody. Goldbodies can specifically recognize the corresponding antigens like the original natural antibodies do, but direct structural evidence for the refolding or restoration of native conformation of the grafted CDRs on AuNPs is still missing and in high demand. Herein we design a new Goldbody that targets an epitope on the lysozyme different from that of the previous antilysozyme Goldbody, and the one circle of helix in the CDR makes it possible to distinguish the unfolded conformation of the free CDR and its folded conformation on AuNPs by circular dichroism (CD) spectroscopy. The refolding of flexible protein fragments on NPs provides unique evidence and inspiration for understanding the fundamental principles of protein folding.

PEGylation of Goldbody: PEG-aided conformational engineering of peptides on gold nanoparticles

“…Accumulating evidence shows that “Goldization” will become a general technique to produce more and more Goldbodies targeting various antigens. 9–11,45,46 And now we show here that the PEGylation of Goldbodies can dramatically reduce the cost of Goldbodies. It is foreseeable that PEGylation would likely become a standard procedure for Goldbodies for their future potential applications in various fields.…”

Biotinylated Au Nanoparticle-Based Artificial Antibody for Detection of Lysozyme by the Lateral Flow Immunoassay and Enzyme-Linked Immunosorbent Assay

“…Second, the surface of “bare” AuNPs can strongly bind with almost any proteins, , and thus the surface of AuNPs has to be functionalized with Pep1 or other hydrophilic molecules to prevent this non-specific binding. As estimated previously, the minimum surface coverage to reduce the strong non-specific binding of AuNPs is about one-fourth of the optimal density for restoring the native conformation of Pep1. , To fill these two roles, we used HS-PEG-SH to passivate the large bare surface of AuNPs left by reducing Pep1 and optimized the density of PEG on AuNPs to help the restoration of the native conformation of Pep1 by restriction of the movement (conformational space) of Pep1 on AuNPs to achieve a favorable entropic effect. ,,, …”

“…These inhibition results are also confirmed by the binding of the AuNP- x PEG/biotin/Pep1 and AuNP- x PEG/biotin/Pep1s with lysozyme as measured by SPR experiments (Figure B and Figure S4), which also show that the interaction between the negative control (AuNP- x PEG/biotin/Pep1s) and lysozyme becomes weaker with the increase of the PEG coverage on the surface of AuNPs, and 500 PEGs per AuNP (14 nm) is the optimal number to assist the restoration of the native conformation of Pep1 (as reflected by the binding and inhibition of lysozyme). Thus, the optimal number of PEGs plays the same role as the optimal number of Pep1 in helping Pep1 restore its native conformation, likely by imposing a suitable restriction on the movement of the reduced number of Pep1 on AuNPs to achieve a favorable entropic effect. ,,, Too low coverage of PEG will give Pep1 too much freedom to move on the surface of AuNPs and to adopt a large number of different conformations; thus, Pep1 cannot maintain its native conformation on AuNPs. On the other hand, an excess amount of PEG will make the surface of AuNPs too crowded, thus hindering the formation of the native conformation of Pep1.…”

“…26,28 To fill these two roles, we used HS-PEG-SH to passivate the large bare surface of AuNPs left by reducing Pep1 and optimized the density of PEG on AuNPs to help the restoration of the native conformation of Pep1 by restriction of the movement (conformational space) of Pep1 on AuNPs to achieve a favorable entropic effect. 26,28,42,43 Pep1 is adapted from the CDR3 fragment of a monoclonal anti-lysozyme antibody, cAb-Lys3, with a Cys residue at its both terminals, so Pep1 conjugates to AuNPs with two Au−S bonds, which is critical to restoring its native conformation on AuNPs. 26,44 The HS-PEG-SH, like Pep1, was also anchored to AuNPs through two Au−S bonds, and it could passivate the bare AuNP surface and help Pep1 to form the correct conformation.…”

“…Thus, the optimal number of PEGs plays the same role as the optimal number of Pep1 in helping Pep1 restore its native conformation, likely by imposing a suitable restriction on the movement of the reduced number of Pep1 on AuNPs to achieve a favorable entropic effect. 26,28,42,43 Too low coverage of PEG will give Pep1 too much freedom to move on the surface of AuNPs and to adopt a large number of different conformations; thus, Pep1 cannot maintain its native conformation on AuNPs. On the other hand, an excess amount of PEG will make the surface of AuNPs too crowded, thus hindering the formation of the native conformation of Pep1.…”