Substrate Binding Tunes Conformational Flexibility and Kinetic Stability of an Amino Acid Antiporter

Abstract

We used single molecule dynamic force spectroscopy to unfold individual serine/threonine antiporters SteT from Bacillus subtilis. The unfolding force patterns revealed interactions and energy barriers that stabilized structural segments of SteT. Substrate binding did not establish strong localized interactions but appeared to be facilitated by the formation of weak interactions with several structural segments. Upon substrate binding, all energy barriers of the antiporter changed thereby describing the transition from brittle mechanical properties of SteT in the unbound state to structurally flexible conformations in the substrate-bound state. The lifetime of the unbound state was much shorter than that of the substrate-bound state. This leads to the conclusion that the unbound state of SteT shows a reduced conformational flexibility to facilitate specific substrate binding and a reduced kinetic stability to enable rapid switching to the bound state. In contrast, the bound state of SteT showed an increased conformational flexibility and kinetic stability such as required to enable transport of substrate across the cell membrane. This result supports the working model of antiporters in which alternate substrate access from one to the other membrane surface occurs in the substrate-bound state.

Cholesterol increases kinetic, energetic, and mechanical stability of the human β2-adrenergic receptor

“…As theoretically predicted (46,47) and experimentally verified using membrane proteins (35,36,(48)(49)(50)(51), increasing the loading rate increases the unfolding forces. We observed a linear relationship between the most probable unfolding force and the logarithm of the loading rate for every interaction.…”

“…Measuring the kinetic response of the individual structural elements by dynamic SMFS (DFS) allows quantification of their unfolding free energy, kinetic stability, conformational variability, and mechanical flexibility (33). Both SMFS and DFS have been applied to characterize these parameters of various membrane proteins and follow how they change on changing their functional state or physiological environment (34)(35)(36)(37)(38)(39).…”

Locating an extracellular K+-dependent interaction site that modulates betaine-binding of the Na+-coupled betaine symporter BetP

“…Quantifying the average interaction strength for a wide variety of loading rates allows estimating the width and the height of the free unfolding energy barriers (Fig. S4), and how they determine the kinetic and mechanical properties of the structural region stabilized (22,33). To investigate the effect of betaine-and K þ -binding on the kinetic and mechanical properties of BetP, we unfolded single BetP molecules at pulling velocities of 100, 300, 600, 1,200, 2,400, and 5;000 nm∕s (Fig.…”

“…In the past, single-molecule force spectroscopy (SMFS) was applied successfully to several transmembrane α-helical and β-barrel membrane proteins to quantify their molecular interactions and locate these onto the primary, secondary, and tertiary structure (16)(17)(18)(19)(20)(21)(22)(23)(24). Dynamic SMFS (DFS) probes these interactions at different force-loading rates and reveals their kinetic parameters (25,26).…”

Investigation of the Interaction between a Bivalent Aptamer and Thrombin by AFM

“…35,37 Quantifying the average interaction strength for a wide variety of loading rates allows us to estimate the width and height of the free unfolding energy barriers and how they determine the kinetic and mechanical properties of the structural region. 38,39 To investigate further the effect of each Bi-8S 0 component on the kinetic and mechanical properties of thrombinÀaptamer binding, we analyzed the dynamic force spectrum (DFS) at pulling speeds of 100, 300, 600, 900, 1200, 2400, and 5000 nm/s in the buffer solutions discussed previously ( Figure S1) and located the most prominent energy barriers along the reaction coordinate.…”