New Method Provides Insights into Protein Shape Changes

Scientists study the shapes and movements of large molecules like proteins to understand how they work in living things. To get very detailed pictures of these molecules, they often use special techniques when the molecules are frozen, crystallized, or dissolved in liquid. This article discusses a new method that can measure the distances between parts of proteins that change shape when they bind with other molecules.

#Methods for Studying Molecules

Traditionally, scientists use methods like X-ray crystallography, cryo-electron microscopy, and nuclear magnetic resonance (NMR) to gather data on the structures of proteins. Each of these methods has its strengths and weaknesses. For instance, X-ray crystallography provides excellent detail but requires the molecules to form crystals. Cryo-EM is good for frozen samples but can struggle with smaller molecules. NMR allows scientists to study proteins in solution but has limits on the distances it can measure.

By measuring the distances between certain points in these proteins, scientists can learn how they change shape and how they interact with other molecules. Techniques like PELDOR/DEER and single-molecule Förster resonance energy transfer (smFRET) help find these distances, but they come with challenges. The new approach we talk about here, called anomalous X-ray Scattering interferometry (AXSI), aims to address some of these challenges.

#Understanding AXSI

AXSI uses X-ray scattering to measure distances between gold labels attached to proteins. The technique takes advantage of how X-rays interact differently with materials based on their atomic structure. By measuring how X-rays scatter when they hit a sample, scientists can infer how far apart the gold labels are, which provides information about the structure of the protein.

One strength of AXSI is that it can measure absolute distances very accurately, and it works even for longer distances. This makes it a powerful tool for studying proteins as they change shape in response to different conditions, such as binding with other molecules.

#Application to Male Protein

In our study, we focused on a protein called MalE, which is part of a system in E. coli that imports maltose, a type of sugar. MalE changes shape when maltose binds to it. This conformational change is crucial for its function.

To use AXSI, we first labeled two versions of MalE with gold nanoparticles at specific positions. Since MalE does not have any natural cysteine residues that can be modified, we created mutant versions of the protein that included two cysteines at chosen locations. These cysteines allowed us to attach the gold particles specifically.

We then measured the scattering of X-rays from these labeled proteins. Using AXSI, we could determine the distances between the gold labels in both the unbound (apo) and bound (holo) states of MalE when maltose was present.

#SAXS and ASAXS Measurements

First, we carried out small-angle X-ray scattering (SAXS) on the unlabeled MalE protein to check the quality of our samples. SAXS provides information about the size and shape of proteins. Then, we performed anomalous small-angle X-ray scattering (ASAXS) on the double-labeled MalE variants by using different X-ray energy levels that are sensitive to the gold labels.

The ASAXS data showed that the distances between gold labels varied when maltose was added. This indicated that the protein was indeed changing shape upon binding with the sugar.

#Analyzing Distance Data

We analyzed the distance data obtained from ASAXS to create a distribution of distances between the gold labels. We found a clear main peak in the distribution, indicating a precise measurement of the distance. The measurements were consistent across different tests, confirming the accuracy of the method.

By comparing the distances measured with AXSI to those obtained from smFRET, we found a strong agreement. This suggests that AXSI is a reliable method for measuring distances in proteins, even when they undergo conformational changes.

#Benefits of AXSI

AXSI offers several advantages over traditional techniques:

1. High Precision: AXSI can measure distances with an accuracy of less than 1 Ångström.
2. Full Distribution: Unlike smFRET, which provides only average distances, AXSI allows for the measurement of the entire distribution of distances, revealing more detailed information about the molecular structure.
3. Broad Applicability: AXSI can be applied to various protein types and conditions, making it a versatile tool in structural biology.

#Future Implications

The success we achieved with AXSI in measuring distances in MalE opens doors for further advancements. This method can be applied to study not only the conformational changes in proteins but also interactions between different proteins, binding kinetics, and other biological processes.

As researchers refine labeling techniques and improve sample preparation, the potential for AXSI as a general tool in structural biology will increase. Additionally, combining AXSI with computer modeling could lead to new insights and better predictions about how proteins function in living organisms.

#Conclusion

In summary, AXSI is a promising new method for measuring distances in proteins, particularly those that change shape. Our studies on MalE demonstrated that AXSI could accurately measure intramolecular distances and provide crucial insights into protein dynamics. As this technique develops, it could play a significant role in enhancing our understanding of protein structure and function in biology.