Molecular mechanism research is a Fascinating area involving multidisciplinary scientists. This study offers insight into the characterization of protein interactions, which is an important subject of this research. The discussion covers case studies on the principles associated with neurotransmitter release and associated factors. The concentration on methods like Nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, as well as other biophysical methods, are also included. These studies have revealed a complex cascading process leading to neurotransmitter release: (i) a tight complex of the soluble SNAP receptor (SNARE) proteins brings the plasma membranes and synaptic vesicle together, essential activity for membrane fusion; (ii) auto-inhibitory closed conformation is adopted by SNARE syntaxin-1 (iii) Munc18-1 plays a vital role by interacting with closed syntaxin-1 and with the SNARE complex; (iv) opening of syntaxin-1 is mediated by Munc13s; (v) through distinct interactions with the SNARE complex, complexions play dual roles; (vi), synaptotagmin-1 interacts simultaneously with the membranes and the SNAREs and acts as Ca21 sensor; and (vii) neurotransmitter release is modulated by a Munc13 homo-dimer to Munc13-RIM hetero-dimer switch. This review highlights the complex molecular mechanisms and explains how these mechanisms are significantly influenced by strong and weak protein interactions.