Coexistence of multiple minor states of fatty acid binding protein and their functional relevance.

Binhan Yu,Daiwen Yang

doi:10.1038/srep34171

Abstract

Proteins are dynamic over a wide range of timescales, but determining the number of distinct dynamic processes and identifying functionally relevant dynamics are still challenging. Here we present the study on human intestinal fatty acid binding protein (hIFABP) using a novel analysis of 15N relaxation dispersion (RD) and chemical shift saturation transfer (CEST) experiments. Through combined analysis of the two types of experiments, we found that hIFABP exists in a four-state equilibrium in which three minor states interconvert directly with the major state. According to conversion rates from the major “closed” state to minor states, these minor states are irrelevant to the function of fatty acid transport. Based on chemical shifts of the minor states which could not be determined from RD data alone but were extracted from a combined analysis of RD and CEST data, we found that all the minor states are native-like. This conclusion is further supported by hydrogen-deuterium exchange experiments. Direct conversions between the native state and native-like intermediate states may suggest parallel multitrack unfolding/folding pathways of hIFABP. Moreover, hydrogen-deuterium exchange data indicate the existence of another locally unfolded minor state that is relevant to the fatty acid entry process.

Highlights

Absence of ligands exists in four conformational states: one major state and three minor intermediate states
One hundred and five 15N-1H HSQC peaks from 130 backbone amides were well resolved in the spectra of hIFABP recorded on both 500 and 800 MHz Nuclear magnetic resonance (NMR) spectrometers. 15N CEST and RD data were analyzed for determining the number of conformational states and kinetic parameters
When the CEST profiles with two dips separated by ≥160 Hz were fitted individually to a two-state exchange model (N ↔ I1, where N and I1 represent native and minor states, respectively), the resultant average exchange rate between N and I1 states and average population of state I1 (PI1) were about 60 s−1 and 3%, respectively

Summary

Introduction

Absence of ligands exists in four conformational states: one major state and three minor intermediate states. The three minor states are native-like and each interconvert with the major state with exchange rates ranging from 36 s−1 to 3086 s−1, suggesting the presence of multiple independent protein folding pathways. A locally unfolded state likely exists in WT hIFABP since the hydrogen-deuterium exchange protection factors of the amides in the second helix are smaller than 100 and those of V17-M21 in the first helix are larger than 10000

Methods

Results

Conclusion