Cytochrome-c (cyt-c), a multi-functional protein, plays a significant role in the electron transport chain, and thus is indispensable in the energy-production process. Besides being an important component in apoptosis, it detoxifies reactive oxygen species. Two hundred and eighty-five complete amino acid sequences of cyt-c from different species are known. Sequence analysis suggests that the number of amino acid residues in most mitochondrial cyts-c is in the range 104 ± 10, and amino acid residues at only few positions are highly conserved throughout evolution. These highly conserved residues are Cys14, Cys17, His18, Gly29, Pro30, Gly41, Asn52, Trp59, Tyr67, Leu68, Pro71, Pro76, Thr78, Met80, and Phe82. These are also known as "key residues", which contribute significantly to the structure, function, folding, and stability of cyt-c. The three-dimensional structure of cyt-c from ten eukaryotic species have been determined using X-ray diffraction studies. Structure analysis suggests that the tertiary structure of cyt-c is almost preserved along the evolutionary scale. Furthermore, residues of N/C-terminal helices Gly6, Phe10, Leu94, and Tyr97 interact with each other in a specific manner, forming an evolutionary conserved interface. To understand the role of evolutionary conserved residues on structure, stability, and function, numerous studies have been performed in which these residues were substituted with different amino acids. In these studies, structure deals with the effect of mutation on secondary and tertiary structure measured by spectroscopic techniques; stability deals with the effect of mutation on T m (midpoint of heat denaturation), ∆G D (Gibbs free energy change on denaturation) and folding; and function deals with the effect of mutation on electron transport, apoptosis, cell growth, and protein expression. In this review, we have compiled all these studies at one place. This compilation will be useful to biochemists and biophysicists interested in understanding the importance of conservation of certain residues throughout the evolution in preserving the structure, function, and stability in proteins.

Proteins are major components of the biological functions in a cell. Biology demands that a protein must fold into its stable three-dimensional structure to become functional. In an unfavorable cellular environment, protein may get misfolded resulting in its aggregation. These conformational disorders are directly related to the tissue damage resulting in cellular dysfunction giving rise to different diseases. This way, several neurodegenerative diseases such as Alzheimer, Parkinson Huntington diseases and amyotrophic lateral sclerosis are caused. Misfolding of the protein is prevented by innate molecular chaperones of different classes. It is envisaged that work on this line is likely to translate the knowledge into the development of possible strategies for early diagnosis and efficient management of such related human diseases. The present review deals with the human neurodegenerative diseases caused due to the protein misfolding highlighting pathomechanisms and therapeutic intervention.

A sequence alignment of yeast cytochrome-c (y-cyt-c) with mammalian cyts-c shows that the yeast protein has a five residue long N-terminal extension. A question arises: Does this N-terminal extension play any roles in the stability, structure, and folding of the yeast protein? To answer this question, in silico and in vitro studies were carried out on the wild type (WT) protein and its five deletants (Δ(-5/-5), Δ(-5/-4), Δ(-5/-3), Δ(-5/-2), and Δ(-5/-1) where Δ denotes the deletion and the numbers refer to the residues deleted, e.g. Δ(-5/-1) denotes the deletion of residues numbered from -5 to -1 (TEFKA), while Δ(-5/-2) denotes the deletion of resides numbered from -5 to -2 (TEFK) and so on). The main conclusion of the in silico study is that the order of stability of deletants and WT protein is Δ(-5/-4) > WT > Δ(-5/-3) > Δ(-5/-5) > Δ(-5/-1) ~ Δ(-5/-2). In vitro studies involved (i) measurements of thermodynamic stability of all proteins by differential scanning calorimetry and from sigmoidal curves of two different structural properties ([θ]222, a probe for detecting change in secondary structure, and Δε405, a probe for detecting alteration in the heme environment), and (ii) characterization of all proteins by various spectral properties. The main conclusions of the in vitro studies are as follows: (i) The order of thermodynamic stability of all proteins is in excellent agreement with that predicted by in silico studies, and (ii) A sequential deletion of the N-terminal extension has no effects on protein structure and folding.

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A sequence alignment of mammalian cytochromes c with yeast iso-1-cytochrome c (y-cyt-c) shows that the yeast protein contains five extra N-terminal residues. We have been interested in understanding the question: What is the role of these five extra N-terminal residues in folding and stability of the protein? To answer this question we have prepared five deletants of y-cyt-c by sequentially removing these extra residues. During our studies on the wild type (WT) protein and its deletants, we observed that the amount of secondary structure in the guanidinium chloride (GdmCl)-induced denatured (D) state of each protein is different from that of the heat-induced denatured (H) state. This finding is confirmed by the observation of an additional cooperative transition curve of optical properties between H and D states on the addition of different concentrations of GdmCl to the already heat denatured WT y-cyt-c and its deletants at pH 6.0 and 68°C. For each protein, analysis of transition curves representing processes, native (N) state ↔ D state, N state ↔ H state, and H state ↔ D state, was done to obtain Gibbs free energy changes associated with all the three processes. This analysis showed that, for each protein, thermodynamic cycle accommodates Gibbs free energies associated with transitions between N and D states, N and H states, and H and D states, the characteristics required for a thermodynamic function. All these experimental observations have been supported by our molecular dynamics simulation studies.

Yeast iso-1-cytochrome c (y-cyt-c) has five extra residues at N-terminus in comparison to the horse cytochrome c. These residues are numbered as -5 to -1. Here, these extra residues are sequentially removed from y-cyt-c to establish their role in folding and stability of the protein. We performed urea-induced denaturation of wild-type (WT) y-cyt-c and its deletants. Denaturation was followed by observing change in Δε405 (probe for measuring change in the heme environment within the protein), [θ]405 (probe for measuring the change in Phe82 and Met80 axial bonding), [θ]222 (probe for measuring change in secondary structure) and [θ]416 (probe for measuring change in the heme-methionine environment). The urea-induced reversible denaturation curves were used to estimate Δ[Formula: see text], the value of Gibbs free energy change (ΔGD) in the absence of urea; Cm, the midpoint of the denaturation curve, i.e. molar urea concentration ([urea]) at which ΔGD = 0; and m, the slope (=∂ΔGD/∂[urea]). Our in vitro results clearly show that except Δ(-5/-4) all deletants are less stable than WT protein. Coincidence of normalized transition curves of all physical properties suggests that unfolding/refolding of WT protein and its deletants is a two-state process. To confirm our in vitro observations, we performed 40 ns MD simulation of both WT y-cyt-c and its deletants. MD simulation results clearly show that extra N-terminal residues play a role in stability but not in folding of the protein.

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