Figure 6. The fluctuation curves of numbers of local folding shapes for PDCD1_MOUSE (red) and PDCD1_HUMAN (blue) proteins. The horizontal axis is the ruler of sequence, and vertical axis the number of local folding variations.
Disorder and flexibility in protein
The nature of protein conformation in biological system is not static, it is flexible following time being and condition change. The process involves intermediate folding states and stable folding states during their synthesis and degradation. These protein folding states may have various half-lifetime, and keep reversible disorder-order transitions. Also, the intrinsically disordered folds indeed exist in some of fragments or entire protein.11Dunker AK, Lawson JD, Brown CJ, Williams RM, Romero P, Oh JS, Oldfield CJ, Campen AM, Ratliff CM, Hipps KW, Ausio J, Nissen MS, Reeves R, Kang C, Kissinger CR, Bailey RW, Griswold MD, Chiu W, Garner EC, Obradovic Z, ”Intrinsically disordered protein”. Journal of Molecular Graphics & Modelling. 19 (1): 26–59, (2001).,22Dyson HJ, Wright PE, ”Intrinsically unstructured proteins and their functions”. Nature Reviews. Molecular Cell Biology. 6 (3): 197–208, (2005)..,33Dunker AK, Silman I, Uversky VN, Sussman JL, ”Function and structure of inherently disordered proteins”. Current Opinion in Structural Biology. 18 (6): 756–64, (2008).
The results from experimental measurement are actually affected by intrinsically disorder and flexibility in protein. About two-thirds of data in PDB do not have the complete 3D structures covering entire sequence by X-ray crystallography measurement because of the unobserved regions that frequently correspond to the disorder.44Le Gall T, Romero PR, Cortese MS, Uversky VN,Dunker AK. Intrinsic disorder in the Protein Data Bank. J Biomol Struct Dyn; 24:325-42, (2007). With nuclear magnetic resonance (NMR) spectroscopy, it can reveal an ensemble of protein structures with flexible dynamics or disorder states limited around a static state. With transmission electron cryomicroscopy (CryoTEM), it can provide new insights for protein structure with large assemblies which allow observation of protein structures in their native environment at cryogenic temperature. With computational simulations, it can obtain protein dynamics trajectories which reveal the disorder and flexibility within protein structure. The protein folding occur in many steps, and it may spend nearly 96% time in some states,  and also in various intermediate states with minimum thermodynamic free energies in energy landscape.55Heath Ecroyd; John A. Carver, ”Unraveling the mysteries of protein folding and misfolding”. IUBMB Life (review). 60 (12): 769–774, (2008). ,66Robert B Best, ”Atomistic molecular simulations of protein folding”. Current Opinion in Structural Biology (review). 22 (1): 52–61, (2012).
Under physiological condition, the protein flexibility substantially plays important role for biological functions. Even after self-assemble into a native state with active function, some parts of proteins may remain folding variations.77Berg JM, Tymoczko JL, Stryer L, ”3. Protein Structure and Function”. Biochemistry. San Francisco: W. H. Freeman. ISBN 0-7167-4684-0, (2002).  The protein biological functions associate with either the native stable structure or the dynamics motion in structure. It is undeniable that the protein structures with both dynamic disorder conformations and stable conformations subsequently are linked to important functions such as allosteric regulation and enzyme catalysis.88Bu Z, Callaway DJ, ”Proteins move! Protein dynamics and long-range allostery in cell signaling”. Advances in Protein Chemistry and Structural Biology. Advances in Protein Chemistry and Structural Biology. 83: 163–221, (2011).,99Kamerlin SC, Warshel A, ”At the dawn of the 21st century: Is dynamics the missing link for understanding enzyme catalysis?”. Proteins. 78 (6): 1339–75, (2010).   Although the so-called native conformation is essential to a specific function in cell, the environmental factors, ligands and other proteins may cause the native conformation into altered folds which may trigger to act different biological function, such as active or inhibiting function or toxic affection. This is actual process when a drug targets proteins to make the conformation changes and to cause activation or inhibition for protein function. Overall, a complete ensemble of conformations really represents the nature of protein folding for multiple functions in physiological condition or various environments. It is significant that the PFVM provides the comprehensive local folding variations for discovery of various conformations in astronomical number. Of course, the remaining question is how to distinguish local folding variations in PFVM with association of function changes. Ultimately, we need better experimental data and further data mining to understand the role of each local folding variation in biological functions.
CONCLUSION
The protein structure fingerprint provided a significant means to probe the protein folding problem, especially overcame the obstacles how to reveal and handle an astronomical number of folding conformations. The folding shapes of 5 amino acid residues, as a universal element, are comprehended, and then expanded over sequence to expose an astronomical number of folding conformations. A set of 27 PFSC alphabetical letters are represented as digital expression. The local folding variations in PFVM really uncovered how the protein folds are correlated by the order of amino acids in sequence. Also, it provides the prospect to construct all possible conformations with astronomical number as well as to predict the most probable conformations for a protein. These advantages information may promote the research in protein structures, such as the protein folding, protein structure prediction, intrinsically disordered protein, protein mutation and protein design etc.