Protein Characterization in 3D: Size, Folding and Functional Assessment in a Unified Approach

TY - JOUR

T1 - Protein Characterization in 3D

T2 - Size, Folding and Functional Assessment in a Unified Approach

AU - Pedersen, Morten Enghave

AU - Gad, Sarah Isbrandt

AU - Ostergaard, Jesper

AU - Jensen, Henrik

PY - 2019/4/16

Y1 - 2019/4/16

N2 - Assessment of protein stability and function is key to the understanding of biological systems and plays an important role in the development of protein-based drugs. In this work, we introduce an integrated approach based on Taylor Dispersion Analysis (TDA), Flow Induced Dispersion Analysis (FIDA) and in-line intrinsic fluorescence which enables rapid and detailed assessment of protein stability and unfolding. We demonstrate that the new platform is able to efficiently characterize chemically induced protein unfolding of Human Serum Albumin (HSA) in great detail. The combined platform enables local structural changes to be probed by monitoring changes in intrinsic fluorescence and loss of binding of a low-molecular weight ligand. Simultaneously, the size of the unfolding HSA is obtained by TDA on the same samples. The integration of the methodologies enables a fully auto-mated characterization of HSA using only a few hundred nanoliters of sample. We envision that the presented methodology will find applications in fundamental biophysics and biology as well as in stability screens of protein-based drug candidates.

AB - Assessment of protein stability and function is key to the understanding of biological systems and plays an important role in the development of protein-based drugs. In this work, we introduce an integrated approach based on Taylor Dispersion Analysis (TDA), Flow Induced Dispersion Analysis (FIDA) and in-line intrinsic fluorescence which enables rapid and detailed assessment of protein stability and unfolding. We demonstrate that the new platform is able to efficiently characterize chemically induced protein unfolding of Human Serum Albumin (HSA) in great detail. The combined platform enables local structural changes to be probed by monitoring changes in intrinsic fluorescence and loss of binding of a low-molecular weight ligand. Simultaneously, the size of the unfolding HSA is obtained by TDA on the same samples. The integration of the methodologies enables a fully auto-mated characterization of HSA using only a few hundred nanoliters of sample. We envision that the presented methodology will find applications in fundamental biophysics and biology as well as in stability screens of protein-based drug candidates.

U2 - 10.1021/acs.analchem.9b00537

DO - 10.1021/acs.analchem.9b00537

M3 - Journal article

C2 - 30916933

SN - 0003-2700

VL - 91

SP - 4975

EP - 4979

JO - Analytical Chemistry

JF - Analytical Chemistry

IS - 8

ER -