The Protein Is Quicker Than the Eye, but Not the AFM

A specially modified atomic force microscope (AFM, top) was used to pull on and unfold a protein embedded in a membrane (bottom). Changes in protein structure bend the AFM tip, as detected by the red laser. Researchers measured many previously hidden changes in the protein’s three-dimensional structure. For example, measurements of just one small part of the protein (right) revealed unfolding and refolding between two intermediate states (dashed lines) over less than a millisecond. [JILA]

Now you see it. Now you don’t. As far as a team of biophysicists at JILA is concerned, the “it” is a membrane protein’s folding behavior. When membrane proteins fold, they form a sequence of intermediate states, more than we can see using conventional techniques such as single-molecule force spectroscopy (SMFS). But the JILA scientists had a trick up their sleeves—they combined SMFS with atomic force microscopy (AFM). The new approach allowed them to capture the protein’s folding steps at microsecond resolution.

The JILA researchers discovered many previously unknown intermediate states by using their apparatus to unfold a protein called bacteriorhodopsin. For example, the JILA team identified 14 intermediate states—seven times as many as previously observed—in just one part of bacteriorhodopsin, a protein in microbes that converts light to chemical energy and is widely studied in research.

This finding is especially significant because it reveals previously unseen behavior of a membrane protein. As a class, membrane proteins are much larger and more difficult to study than globular proteins, which are the focus of most protein folding research. Yet membrane proteins are of intense interest, too, because they are involved in many human diseases and are the targets of many medicinal drugs.

Knowledge of protein folding is important because proteins must assume the correct three-dimensional structure to function properly. Misfolding may inactivate a protein or make it toxic.

By showing how the folding of membrane proteins can be studied in more detail, the JILA scientists have shown how researchers may better understand previously obscure biophysical processes related to diseases such as neurodegeneration and cancer.

“If you miss most of the intermediate states, then you don't really understand the system,” said Tom Perkins, Ph.D., the leader of the team at JILA, which is a partnership between the National Institute of Standards and Technology (NIST) and the University of Colorado, Boulder. “The increased complexity [we uncovered] was stunning. Better instruments revealed all sorts of hidden dynamics that were obscured over the last 17 years when using conventional technology.”

Details of the new work appeared March 3 in the journal Science, in an article entitled “Hidden Dynamics in the Unfolding of Individual Bacteriorhodopsin Proteins.”

“Using force spectroscopy optimized for 1-microsecond resolution, we reexamined the unfolding of individual bacteriorhodopsin molecules in native lipid bilayers,” wrote the article’s authors. “The experimental data reveal the unfolding pathway in unprecedented detail. Numerous newly detected intermediates—many separated by as few as two or three amino acids—exhibited complex dynamics, including frequent refolding and state occupancies of <10 μs.”

The JILA scientists used an AFM to stretch bacteriorhodopsin and measure its extension (in nanometers) at various pulling speeds (measured in nanometers per second). The new measurements were made possible by JILA's prior development of short, soft AFM probes, which quickly gauge abrupt changes in force—signaling an intermediate state—as a protein unfolds. Further refinements of these probes allowed JILA researchers to probe bacteriorhodopsin 100 times faster (in 1 μs) and with 10 times the precision in pulling force of prior work.

The JILA team found that intermediate states were not only more numerous than expected but also lasted as little as 8 μs. The findings resolved long-standing discrepancies between past experimental data and molecular simulations, giving confidence to using such simulations to further probe the behavior of membrane proteins.

The JILA team's discovery and techniques could be applied to many other molecular studies, including those of medical interest such as interactions between proteins and medications.