Investigating Lipase/Stain Interactions: Determining Interfacial Protein Conformation with Surface Spectroscopy

TY - JOUR

T1 - Investigating Lipase/Stain Interactions

T2 - Determining Interfacial Protein Conformation with Surface Spectroscopy

AU - Saeed, Khezar H

AU - Strunge, Kris

AU - Pedersen, Kasper B

AU - Truelsen, Sigurd F

AU - Christensen, Sune M

AU - Olsen, Lars

AU - Schiøtt, Birgit

AU - Weidner, Tobias

PY - 2024/8/29

Y1 - 2024/8/29

N2 - Conventional bulk protein structure determination methods are not suitable for understanding the distinct and diverse interactions of proteins with interfaces. Notably, interfacial activation is a feature common to many lipases involving movement of a helical "lid" region upon contact with a hydrophobic surface to expose the catalytic site. Here we use the surface specificity of vibrational sum frequency generation spectroscopy (VSFG) spectroscopy to directly probe the conformation of Thermomyces lanuginosus lipase (TLL) at hydrophobic interfaces. The TLL-catalyzed reaction at the air/water interface is monitored by VSFG spectroscopy, showing loss of ester carbonyl modes and appearance of carboxylate stretching modes of the fatty acid products. Furthermore, comparison of experimental and calculated VSFG spectra of the amide I band of TLL allows us to discern the subtle structural changes involved with lid-opening at a hydrophobic surface. Finally, we report a likely orientation of this lid-open state, which interacts with the surface through a loop region away from the lid and active site. This experimental framework for probing protein structure and function at interfaces addresses a significant problem in protein science that is not only impeding the design of better enzymes for biotechnology applications but also drug discovery targeting membrane associated proteins.

AB - Conventional bulk protein structure determination methods are not suitable for understanding the distinct and diverse interactions of proteins with interfaces. Notably, interfacial activation is a feature common to many lipases involving movement of a helical "lid" region upon contact with a hydrophobic surface to expose the catalytic site. Here we use the surface specificity of vibrational sum frequency generation spectroscopy (VSFG) spectroscopy to directly probe the conformation of Thermomyces lanuginosus lipase (TLL) at hydrophobic interfaces. The TLL-catalyzed reaction at the air/water interface is monitored by VSFG spectroscopy, showing loss of ester carbonyl modes and appearance of carboxylate stretching modes of the fatty acid products. Furthermore, comparison of experimental and calculated VSFG spectra of the amide I band of TLL allows us to discern the subtle structural changes involved with lid-opening at a hydrophobic surface. Finally, we report a likely orientation of this lid-open state, which interacts with the surface through a loop region away from the lid and active site. This experimental framework for probing protein structure and function at interfaces addresses a significant problem in protein science that is not only impeding the design of better enzymes for biotechnology applications but also drug discovery targeting membrane associated proteins.

UR - http://www.scopus.com/inward/record.url?scp=85201697678&partnerID=8YFLogxK

U2 - 10.1021/acs.jpcb.4c03173

DO - 10.1021/acs.jpcb.4c03173

M3 - Journal article

C2 - 39158521

SN - 1520-6106

VL - 128

SP - 8162

EP - 8169

JO - The journal of physical chemistry. B

JF - The journal of physical chemistry. B

IS - 34

ER -