The Experiment: Catching the Spike Protein in Action
When the COVID-19 pandemic emerged, IISc researchers pivoted to confront the new threat. A team led by Dr. Somnath Dutta of the Molecular Biophysics Unit targeted the virus's most recognizable feature—the spike (S) protein that forms the characteristic "crown" on the viral surface 2 7 .
The Critical Question
Previous studies had visualized the spike protein at either acidic or basic pH levels, but its structure "at physiologically relevant conditions—at which the virus actually infects the host cells—remained poorly understood" 2 . This was a critical gap—after all, the human body maintains a precise pH balance, and understanding how the spike protein behaves under these conditions could reveal vulnerabilities in the virus's infection strategy.
Methodological Approach
Dutta's team employed single-particle cryo-electron microscopy (cryo-EM), a revolutionary technique that allows scientists to visualize biological molecules at near-atomic resolution by flash-freezing them in vitreous ice and imaging them with electrons 2 7 . This method preserves the native structure of proteins in ways that other techniques cannot.
| Step |
Procedure |
Purpose |
| Sample Preparation |
Purify viral proteins in solution at specific pH conditions |
To create a representative sample for analysis |
| Vitrification |
Rapid freezing of sample in liquid ethane |
To preserve native structure in amorphous ice |
| Data Collection |
Capture thousands of electron micrograph images |
To obtain 2D projection images of particles in random orientations |
| Image Processing |
Classify particles computationally based on orientation and conformation |
To group similar protein structures for detailed analysis |
| 3D Reconstruction |
Generate 3D density maps from 2D projections |
To visualize the complete three-dimensional structure |
Table 2: Step-by-Step Process of Single-Particle Cryo-EM Analysis
Revelations from the Research
The results were striking. The team discovered that the spike protein exists not as a single static structure, but as a dynamic molecule shifting between multiple conformations 2 7 . Two primary states dominated:
Closed Conformation
The protein is compact, likely hiding its receptor-binding domain.
Open Conformation
The protein is extended, exposing the domain that binds to human ACE2 receptors—the first step in cellular invasion.
Most remarkably, the proportion of open-conformation spikes was highly dependent on pH. At physiological pH (7.4), approximately 68% of spike proteins adopted the open conformation—the form primed for infection. This percentage dropped when the environment became more acidic or basic 2 7 . The virus, it seemed, had evolved to maximize its infectivity under the precise conditions found in the human body.
| pH Level |
Biological Context |
Percentage in Open Conformation |
Infection Potential |
| pH 6.5 (Slightly Acidic) |
Some bodily compartments |
Less than 68% |
Reduced |
| pH 7.4 (Physiological) |
Human blood and tissues |
Approximately 68% |
Optimal |
| pH 8.0 (Slightly Basic) |
Some physiological conditions |
Less than 68% |
Reduced |
Table 3: Percentage of Spike Proteins in Open Conformation at Different pH Levels
Perhaps most importantly, the research revealed that different conformational states had varying binding affinities for neutralizing antibodies 2 7 . This explained why some antibodies were more effective than others at blocking infection and suggested new strategies for vaccine design.
