Protein K

MD simulations of hen egg white lysozyme (HEWL), bovine pancreatic trypsin inhibitor (BPTI), ubiquitin (Ubq), and the B3 domain of Protein G (GB3) were performed using Desmond version 2.1.0.1 and the Amber ff99SB or the modified Amber ff99SB-ILDN force fields. The TIP3P water model20 was used for simulations of HEWL, Ubq, and GB3, and the TIP4P-Ew water model21 (link) was used for simulations of BPTI. Simulation parameters were the same as in the simulations of small helical peptides, apart from the fact that a 64³ PME grid was used for HEWL and a 48³ grid was used for BPTI, Ubq, and GB3. Simulations of HEWL, BPTI, Ubq, and GB3 were initiated from PDB22 (link) entries 6LYT, 5PTI, 1UBQ, and 1P7E solvated in cubic water boxes containing 10,594, 4215, 6080, and 5156 water molecules, respectively. The net charge of the proteins was neutralized with sodium or chloride ions. Each system was initially subject to energy minimization, followed by 1.2 ns of MD simulation in the NPT ensemble during which the temperature was increased linearly from 10 to 300 K, and position restraints on the backbone atoms were annealed from 1.0 to 0.0 kcal mol⁻¹ Å⁻¹. After this initial relaxation, each system was simulated for 6 ns in the NPT ensemble. The frame of this simulation with the volume closest to the average volume was selected as the starting conformation for a production run of 1.2 μs in the NVT ensemble. The trajectories obtained from the NVT runs were used for subsequent data analysis.

Dehydrogenase activity was determined using the method based on the Erdogan Eliuz study (Erdoğan Eliuz, 2021 (link)). Colourless TTC accepted H during cellular respiration and produced insoluble red-coloured 2,3,5-triphenylformazan (TF). The reduction amount of TTC was converted to determine the dehydrogenase activity. The absorbance of TF molecules was measured at 485 nm to determine the activity of dehydrogenase in the cells after adding antimycin A₁ into the culture medium of R. solani.
A test tube containing 10 mL of liquid potato medium was incubated at 180 rpm and 25°C for 4 h. Then, antimycin A₁ was added to final concentrations of 6.66 μg/mL and 13.33 μg/mL, and the culture was continued at 25°C and 180 rpm for 2 h. Distilled water was used as a blank control, and validamycin was used as a positive control. Fifty microlitres of the fungal mixture cultured for 2 h as described above and 50 μL TTC-glucose solution were added to a 96-well plate, and the mixture was shaken at 100 rpm in a shaking incubator for 30 min to form the red substance TF. Finally, the dehydrogenase activity was measured by absorbance at 485 nm using an automatic microplate reader. Dehydrogenase activity (%) = (ODx/ODc) × 100, where ODx and ODc represent the absorbance of treated and control samples, respectively.
Detection of protein leakage was performed as follows: 50 μL of the mixture of R. solani and antimycin A₁ was transferred into a 96-well plate, 50 μL of Coomassie brilliant blue dye reagent was added to the 96-well plate for staining for 5 min, and colour was developed in the 96-well plate. Intracellular protein leakage was determined using the method based on the Erdogan Eliuz study (Erdoğan Eliuz, 2021 (link)). The absorbance was measured at 595 nm with an automatic microplate reader, and the protein leakage rate in the culture medium treated with antimycin A₁ was calculated.
Protein leakage rate (%) = (ODx − ODc)/ODc × 100, where ODx and ODc represent the absorbance of the sample group treated with antimycin A₁ and the blank control group, respectively.
Detection of K⁺ leakage was performed as follows. The KNO₃ standard solution was prepared, 5 mL of the abovementioned mixture of R. solani treated with antimycin A₁ was centrifuged at 10000 × g for 5 min, 0.5 mL of the supernatant was placed in a 50 mL centrifuge tube, 4.5 mL of caesium chloride solution (5 g/L) was added, and then 45 mL of deionized water was added again. The solution was thoroughly mixed, and the absorption value was determined by an atomic absorption spectrophotometer.

This study applied
the fuzzy oil drop (FOD) model.^{14 (link),15 (link)} A short description
is presented here to facilitate the interpretation of the results
obtained by using the model. The basic assumption introduces treating
a protein structure as an effect of the micellization process. Amino
acids are bipolar molecules with a diverse polarity–hydrophobicity
relationship. Bipolar molecules in an aqueous environment form ordered
arrangements with a distribution characterized by a hydrophilic surface,
with hydrophobic residues concentrated in the center. The hydrophobicity
distribution in such a system can be described by a three-dimensional
(3D) Gaussian function spanning the protein molecule. The values of
the parameters (σ_X, σ_Y, and σ_Z; eq 1) are adopted
to the size and shape of the molecule, thereby allowing the representation
of any globular form of the protein molecule.
The value of the
3D Gaussian function
at points representing consecutive amino acids (i.e., positions of the effective atoms, which are the averaged positions
of the atoms comprising a given amino acid) expresses the idealistic
hydrophobicity level referred to in the model as the theoretical T_i (Figure 1A).
In exceptional cases, the structure of the protein
exactly reproduces
a distribution according to a 3D Gaussian function. The actual hydrophobicity
level of a given amino acid results from the intrinsic hydrophobicity
of each amino acid and its interaction with its neighbors (r_ij: distance between effective
atoms). The Levitt function was used for the calculation of the observed
hydrophobicity level (O)^{16 (link)} (eq 2; Figure 1A).
The T and O distributions after
normalization can be subjected to comparative analysis.
The
normalization is expressed by 1/H_sum^T for the T distribution
and 1/H_sum^O for the O distribution. The T_i and O_i are calculated for all
residues. To make them normalized, each of the compounds is divided
by the sum of all T_ij and O_ij. Quantitatively, the compatibility/incompatibility
of the O distribution against the T distribution (reference distribution) is expressed by divergence
entropy introduced by Kullback–Leibler^{17 (link)} (eq 3). where P_i is the distribution analyzed (O distribution
in our model) and Q_i is
the reference distribution (T in the FOD model).
However, the D_KL value (entropy) cannot
be interpreted. Therefore, another reference distribution (R) (Figure 1A) was introduced in which each amino acid represents the same level
of hydrophobicity equal to 1/N, where N is the number of amino acids in the structural unit under consideration.
The D_KL value is determined for the
relationship of the O distribution toward the R distribution (the O|R relationship). A comparison of these two D_KL values indicates the similarity of the O distribution to one of the two reference distributions (T and R). A smaller D_KL value indicates the similarity of the compared distributions.
The relative distance (RD) value of a protein is expressed as follows where RD < 0.5 is interpreted as a protein
with a hydrophobic core (Figure 1B).
A protein composed of amino acids linked
by covalent bonds (peptide
bonds) has limited possibilities (limited mobility) to reproduce the
micelle structure. The degree of adaptation of the O distribution toward the T distribution appears
to vary. Down-hill, fast-folding, ultrafast-folding proteins represent
a status with very low RD values;^{18 (link)} enzymes,
whose structure requires a substrate-binding cavity, show a local
mismatch in the form of local hydrophobicity;^{19 –21 (link)} the complexation
area can be recognized as a local hydrophobicity excess.^{22 (link),23 (link)} The elimination of residues of highest discrepancy between the O_i and T_i values
enables the identification of a moiety fulfilling the condition of
RD < 0.5, indicating that this is responsible for solubility. The
local discrepancies may also be recognized as a potential drug-binding
locus.^{24 (link)}Water is not the only milieu
for protein activity. The exposure
of hydrophobic residues on the surface is required for the stability
of the membrane-anchored protein, which is opposite to the water environment.
This opposite distribution of hydrophobicity in membrane proteins
can be expressed as a function complementing to 1. In practice, the
opposite distribution is determined as follows (Figure 2)
However, the omnipresence of water influences the
form of the hydrophobic
distribution in membrane proteins in the following form
In the formula, the K parameter expresses
the
force with which the field originating from the water environment
is modified by factors other than water, including hydrophobic factors
(Figures 1C and 2).
The graphical presentation of opposite
function and calculation
of K parameter is shown in Figures 1D and 2.
The M distribution is calculated for the minimal
value of D_KL(O|M) (Figures 1C and 2) to find the modified T distribution, possibly the closest one with respect to the O distribution. The M distribution plays
the role of T distribution in a nonaqueous environment
modified by other factors. The description of the protein according
to the final form of the FOD-modified (FOD-M) model is thus expressed
in terms of the RD and K parameter values. The value
of the RD parameter expresses the degree of similarity/dissimilarity
of the O distribution to the T distribution
(automatically the R distribution). The K parameter, however, expresses the extent to which the nonaqueous
environment affects protein structure formation.
Proteins characterized
by low K value s are listed
in.^{25 (link),26 (link)} A protein functioning in the periplasmic
space exhibits a structure described by the parameter value K = 0.6.^{27 (link)} Membrane proteins
(e.g., rhodopsin described by the value K = 0.9) are described by K value s > 1.0.^{28 (link)–32 (link)} The present paper discusses a protein, chaperonin, which can represent
a status with K > 3. A high value of the K parameter is also shown by a protein folded in the external
field environment provided by chaperonin.