In Krammer's theory, stiffness and dissipation coefficient of a protein determine the rate of their conformational change. Using atomic force microscope, it is possible to measure viscoelasticity of a single protein, wherein it's dissipative and elastic nature is directly and independently measured. Such measurements are performed, either by measuring the thermal fluctuations of the protein held under a constant force, or by providing small modulations to the protein by dithering the cantilever and measuring its response. In small amplitude approximation, where dither amplitude is comparable to persistence length of polymers, it is possible to measure the protein's viscoelastic response accurately. We measured dissipation in I27 at extremely low pulling speeds (∼ 50 nm/s) and low dither frequencies (∼ 100 Hz). At these experimental parameters the dissipation is found to be ∼ 10 −5 kg/s, well above the detection limit of conventional AFM and upper limit predicted by Benedetti et al. Our stiffness data clearly reveals unfolding intermediate of titin's individual immunoglobulin units. The intermediate is elongation of folded domains by ∼ 8Å, wherein two hydrogen bonds are broken between beta sheets. It was possible to measure this elongation in our experiments. The directly measured internal friction of unfolded polymer chain shows a scaling with tension on the chain. The measurements show that it is possible to measure internal friction in single molecules unambiguously using small amplitude AFM. It suggests that systematic experiments to unravel the relation between directly measured internal friction and folding rates of proteins are possible. INTRODUCTIONIn last few decades, a number of experimental techniques have been developed to observe and manipulate matter at atomistic and molecular scales [1][2][3][4][5][6][7][8][9][10]. These developments have a lasting impact on the field of molecular biology as well as on molecular nanotechnology. Single macromolecules such as proteins, flexible polymers and polysaccharides are stretched and coil-to-stretched or folded-to-unfolded transitions under the application of external force are routinely observed. The central goal of these experiments is to mimic the molecular response under natural situations such as ligand-receptor binding, allosteric signalling and the stress induced conformational changes. They measure kinetics of unfolding of a protein and binding affinities of ligands to receptors.Along with optical tweezers [3, 9-12], Atomic Force Microscopy has acquired a unique position in this quest due to its unprecedented spatial resolution in physiological conditions [5][6][7][13][14][15][16][17]. In typical AFM experiment, mica or Au substrate is sparsely coated with the biological macromolecule and is placed in the liquid environment. A sharp probe attached to a cantilever spring is then brought close to a molecule. The protein is allowed to attach to it from either C or N terminus through nonspecific binding. The bending in the cantilever beam is measur...