Research Program
Biomolecular Devices and Analysis

Objectives
Biological molecules are commonly viewed as static structures when in fact they constantly undergo thermodynamically-driven conformational fluctuations. These large-scale conformational changes can be essential for biological function, macromolecular association, complex formation and self-assembly. The long term goal of this project is to develop microfabricated tools that monitor both fluctuations and conformational changes of proteins and RNA over a range of biologically-relevant length and time scales. Access to short times is essential to capture fast microsecond fluctuations while access to different length scales will allow us to associate local motions with global changes in size and shape. Our goal is to coordinate conformational dynamics with structural changes.

Methods
A rapid change in solution can generate a large thermodynamic perturbation to dissolved protein or RNA, triggering macromolecular conformational changes like folding, unfolding or misfolding.  Microfabricated mixing devices have been developed that exploit rapid diffusion of small molecules on the micron length scale, resulting in microsecond scale changes in solvent. The mixing efficiency of these devices can be optimized through numerical simulations of flow conditions. Device performance can be verified by control experiments. These devices require miniscule amounts of samples, and with thoughtful design, can be interfaced with different experimental probes. For this project, optically-transparent devices are coupled with optical probes to monitor local conformational changes of molecules with attached fluorescent labels. Silicon-based  microfabricated devices of the same design have been coupled with x-ray scattering techniques to probe global conformational changes. This project involves extensive use of NBTC fabrication and characterization facilities.

Summary
Microfluidic mixers have been developed and applied to study functional conformational dynamics of proteins. In a recently completed study, a 5-inlet channel microfluidic mixer was coupled with multiphoton microscopy to probe the kinetics of conformational changes of the protein calmodulin upon binding of 4 Ca²⁺ ions. In conjunction with conventional stopped-flow mixing devices, the kinetic rates of the conformational changes in two homologous globular domains of calmodulin were measured. A rapid conformational change was discovered on a time scale accessible only to the microfabricated mixer and signaled the presence of a state in which two of four Ca²⁺ binding sites are occupied. The millisecond lifetime of this state suggests a biological role for this transient intermediate.

In the past year, we extended the study of conformational dynamics to other protein and RNA systems. We have identified experimentally accessible conditions that convert the protein apomyoglobin to an amyloid state. Equilibrium structural fluctuations were measured in the microfluidic flow channel using Fluorescence Correlation Spectroscopy (FCS). We also used the microfluidic mixer to probe the rapid collapse of a small RNA fragment, upon addition of Mg²⁺ ions. The collapse time scale of a few microseconds was found to be comparable to fluctuations at equilibrium as determined by FCS. Continuation of these studies is the principal motivating factor of this proposal.

Accomplishments

• An optically transparent microfluidic mixer has been optimized for microsecond scale studies of fluorescently labeled macromolecules
• Conformational dynamics of calmodulin, a ubiquitous calcium sensing protein, were detected following a rapid jump in [Ca²⁺]
• A transient conformational state of calmodulin was detected, with only 50% occupation of the Ca²⁺ binding sites