The thermal stability of barnase has been studied using constant pressure and temperature (CPT) molecular dynamics at different temperatures. Barnase X-ray coordinates were obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB code:1rnb). Simulations were performed at 285, 295, 300, 335, 345, and 395 K in explicit water under periodic boundary conditions for 280 ps. For each simulation, conformations were saved every 0.2 ps. Root mean square deviation (RMSD) values were calculated relative to the starting structure at 300 K and at time t = 0. Root mean square fluctuation (RMSF) values were calculated relative to the average structure obtained from the 300K simulation. Both root mean square deviation and fluctuation analysis indicated the presence of discrete regions of hyper-sensitivity along the barnase polypeptide chain. These regions exhibited spikes in flexibility prior to any global structural changes. The specific changes in barnase backbone flexibility are accompanied by increased phi/psi angle fluctuations. These results suggest the presence of early denaturation sites or denaturation nuclei whose local structure is disrupted prior to global structure disruption. Identification of denaturation nuclei suggests that appropriate amino acid replacements at these sites may lead to the design and development of more stable barnase mutants. This strategy of identifying denaturation nuclei in protein structures may represent a first step in the design of more stable protein structures.