The  ability to pick up  and manipulate single molecules and measure the rupturing force distributions under varying loading rates is utilized in providing  experimental confirmations of many theoretical ideas in non-equilibrium statistical physics . The prominent example is that of Jarzynski's equality. Dynamic AFM  can become a test bed of many recent theoretical ideas in physics with serious implications for unraveling the functioning of single biomolecules. For instance, a time-dependent periodic driving of   fluctuating non equilibrium system produces a phase lag between the response and drive. The  dynamic  AFM has now been successful for the first time in  providing direct  and artefact-free measurements of such a phase lag. Further,  by controlling the speed of forcing, this lag can be reduced or enhanced. The phase lag observed in case of I-27 immunoglobulins  of titin, thus not only gives internal friction coefficient using   the framework of linear response theory, but also can be used to verify some claims in theoretical work carried out so far in this field. 

Some proteins  are known to exhibit functional heterogeneity. This means that there are a host of conformations, all in the ground state, capable of delivering distinct functions.

 This phenomenon is  referred to as structural polymoprphism.  These structures are conformational isomers since it is the same molecule preferring a set of different conformations. These structural heterogeneities were not readily accessible in x-ray crystallography, however with advent of cryo Electron Microscopy, plethora of 3D structures of a given protein are increasingly being reported.  All atom MD simulations are now able to predict many  folding pathways from fully unfolded proteins.  The important aspect of such  conformational isomerism  is the inter-conversion rates between substates. Markov state models do capture the conformational dynamics  

 The role of single molecule approach in understanding such dynamics is   underscored  in a set excellent reviews.  Photoinduced Electron transfer Fluorescence correlation spectroscopy (PET-FCS) provides information about structural fluctuations. Single molecule FRET is able to report  conformational fluctuations. However, the time-scales probed by these optical techniques are in the  range of ps to tens of ns.       It is challenging to map slow interconversion dynamics  experimentally.  Using  interferometer-based  dynamic AFM, one is  able to measure phase lags and thereby average energy lost in each cycle,  as the protein is periodically driven over its many  substates. It is possible to do these measurements over a range of frequencies ( 10 Hz - 10 KHz)  allowing a larger time window at slower times-scales for capturing this dynamics. Further improvement in detection sensitivity will allow stiffer and thereby,  faster cantilevers to expand this window to lower times scales.       This  opens up a possibility of complementary measurement of slow interconversion  rates between substates. To obtain this dynamics under no force conditions,  one needs   models to analyse dynamic AFM data, similar to the ones developed for constant velocity pulling experiments. Another exciting possibility for the future is to combine  single molecule optical spectroscopic  tools to interferometer-based  dynamic AFM  developed by us.

Flexible or mobile domains are part   of  many large  multi-domain proteins and render them  the ability to interact with many partners. This is referred to as promiscuity. A large class  of enzymes, enzyme activators  and receptors exhibit this trait, notably Cytochrom P450,  Calmodulin , PDZ domains  and kinases  Flexibility allows protein domains to take part in different reactions.  In protein interactome, such proteins play a role of  hub  and tend to have intrinsically disordered regions  It is argued that loss of specificity and  enhanced dynamism  is important for  protein function and possesses evolvability.It is also suggested that the flexibility in mobile domains is affected by  presence of other domains in multi-domain proteins. A relatively straightforward proposal would be to map viscoelasticity of known mobile( promiscuous ) domains using dynamic AFM and correlate this information with variation in structural flexibility  observed in superfamilies such as CYP  or PDZ  domains.  Viscoealsticity provides both stiffness and dynamism by reporting the mechanical relaxation or retardation time scales. 

Molecular bonds,  such as those between ligand and receptors, are of the most intriguing kind. They play a key role in all  biological processes. Sometimes they  exhibit counter-intuitive  properties such as becoming stronger under force. They are referred to as catch bonds Catch bonds play a central role in most biological adhesive interactions that has to survive tensile  force on it. The bonds that weaken and eventually break under force are called slip bonds. Many years after the prediction of catch-bonds, their existence was revealed in a pioneering AFM experiment   The mechanism,  by which,  catch-bonds work has not been revealed entirely.  There are many theoretical  models and some key observations about these bonds are reported.  Dynamic AFM offers a crucial advantage of mapping catch bond's viscoelastic response and comparing it with slip bonds. Direct and simultaneous mapping of viscous and elastic component may provide insight into unraveling physics of catch-bonds. 

A  direct  impact  of successful characterization of viscoelastic properties of single proteins is on understanding function  of proteins which are under mechanical tension,  such as  immunoglobulins of titin,  talins ,spectrins  and also mechanosensors such as   Cadherins.  Often,  the tension in the folded state is  responsible for  transmission of  force.  For instance, inside the inner ear,  opening of ion channel to produce action potential as a response to sound waves, involves  modulation of tension  in a cadherin complex called tip-link.  Mapping of  viscoelasticity parameters of  protein domains modelled as soft solids will provide insight into which site-specific mutation is responsible for diseases that might involve softening or hardening of  mechanosenstive proteins. In this sense, dynamic AFM serves as a tool to identify the actual alteration in mechanical properties,  such as its elasticity and viscosity,   due to  mutation.   Early Hearing Loss  and  Cardiomyopathy are two of the examples from, arguably  a  long list of diseases,  which are suspected to be a result of   malfucntioning of mechano-sensitive proteins due to altered mechanical properties.