Configuration-dependent properties of randomly coiling polynucleotide chains. II. The role of the phosphodiester linkage

The dependence of the unperturbed dimensions of randomly coiling polynucleotides on the rotations about the phosphodiester linkages of the chain has been examined in order to understand the conformational discrepancies, set forth in paper I, regarding these angles (ω′ and ω). Large values of the characteristic ratio 〈r²〉₀/nl² , which agree with the experimental behavior of the chain, are obtained only if a sizeable proportion of the polymer residues have trans ω′ values. The asymmetric torsional potential that is believed to arise from gauche effects associated with the P-O bonds has been approximated using a hard core model. The calculated characteristic ratio exhibits a strong dependence upon the magnitude of this torsional barrier (separating trans and gauche conformations) and shows agreement with experimental values for polyribonucleotides only if this energy difference is 1 kcal/mol or less.     

Cooperative transition between two helical conformations in a linear system: Poly-L-proline I ⇌ II. I. Equilibrium studies

The solvent-induced conformational transition between the two helical forms of poly-L -proline is studied as a model for cooperative order ? order transitions. The chain length dependent equilibrium data in two solvent systems are described by Schwarz's theory, which is based upon the most general formulation of the linear Ising model with nearest neighbor interactions. The parameter σ which describes the difficulty of nucleation of a I (II) residue in an uninterrupted II (I)-helix is 10^?5 in both solvent systems. The ratios of the nucleation difficulties of states I and II at the ends of the chains β′ and β″ are very different in the two systems. Nucleation difficulty within the chain is interpreted as being due to unfavorable excess interaction energies at the I–II and II–I junctions, which add up to 7 kcal/mole of nuclei as calculated from the σ value. A similar value is computed from the atomic interactions at the junctions. In contrast to this intrinsic properly of poly-L -proline, the energies of I and II residues at the ends are heavily influenced by interactions of the endgroups with the solvent. The above values of the nucleation parameters are determined by a new least-square fitting procedure which does not necessitate the assumption of the dependence of the equilibrium constant s for propagation upon the external parameters, but yields this function from the experimental transition data. A quantitative explanation of this experimental s function through the binding of solvent is attempted. In the transition region a very small free energy change (about 0.1 kcal/mole), arising from a preferential binding of solvent molecules to one of the conformational states, is sufficient for a complete conversion from one helical form to the other.     

Stability of polypeptide conformational states as determined by computer simulation of the free energy

A method is developed to extract the entropy of polypeptides and proteins from samples of conformations. It is based on techniques suggested previously by Meirovitch, and has the advantage that it can be applied not only to states in which the molecule undergoes harmonic or quasiharmonic conformational fluctuations, but also to the random coil, as well as to mixtures of these extreme states. In order to confine the search to a region of conformational space corresponding to a stable state, the transition probabilities are determined not by “looking to the future,” as in the previous method [H. Meirovitch and H. A. Scheraga (1986) J. Chem. Phys. 84 , 6369–6375], but by analyzing the previous steps in the generation of the chain. The method is applied to a model of decaglycine with rigid geometry, using the potential energy function ECEPP (Empirical Conformational Energy Program for Peptides). The model is simulated with the Metropolis Monte Carlo method to generate samples of conformations in the α-helical and hairpin regions, respectively, at T = 100 K. For the α-helix, the four dihedral angles of the N- and C-terminal residues are found to undergo full rotational variation. The results show that the α-helix is a more stable structure than the hairpin. Both its Helmholtz free energy F and energy E are lower than those of the hairpin by ΔF ～ 0.4 and ΔE ～ 0.3 kcal/mole/residue, respectively. It should be noted that the contribution of the entropy ΔS to ΔF is significant (TΔS ～ 0.1 kcal/mole/residue). Also, the entropy of the α-helix is found to be larger than that of the hairpin. This is a result of the extra entropy arising from the rotational freedom about the four terminal single bonds of the α-helix.     

Prediction of the native conformation of a polypeptide by a statistical-mechanical procedure. III. Probable and average conformations of enkephalin

The program SMAPPS (Statistical-Mechanical Algorithm for Predicting Protein Structure) was originally designed to determine the probable and average backbone (?, ψ) conformations of a polypeptide by the application of equilibrium statistical mechanics in conjunction with an adaptive importance sampling Monte Carlo procedure. In the present paper, the algorithm has been extended to include the variation of all side-chain (χ) and peptide-bond (ω) dihedral angles of a polypeptide during the Monte Carlo search of the conformational space. To test the effectiveness of the generalized algorithm, SMAPPS was used to calculate the probable and average conformations of Met-enkephalin for which all dihedral angles of the pentapeptide were allowed to vary. The total conformational energy for each randomly generated structure of Met-enkephalin was obtained by summing over the interaction energies of all pairs of nonbonded atoms of the whole molecule. The interaction energies were computed by the program ECEPP /2 (Empirical Conformational Energy Program for Peptides). Solvent effects were not included in the computation. The results of the Monte Carlo calculation of the structure of Met-enkephalin indicate that the thermodynamically preferred conformation of the pentapeptide contains a γ-turn involving the three residues Gly²-Gly³-Phe⁴. The γ-turn conformation, however, does not correspond to the structure of lowest conformational energy. Rather, the global minimum-energy conformation, recently determined by a new optimization technique developed in this laboratory, contains a type II′ β-bend that is formed by the interaction of the four residues Gly²-Gly³-Phe⁴-Met⁵. A similar minimum-energy conformation is found by the SMAPPS procedure. The thermodynamically preferred γ-turn structure has a conformational energy of 4.93 kcal/mole higher than the β-bend structure of lowest energy but, because of the inclusion of entropy in the SMAPPS procedure, it is estimated to be ～ 9 kcal/mole lower in free energy. The calculation of the average conformation of Met-enkephalin was repeated until a total of ten independent average conformations were established. As far as the phenylalanine residue of the pentapeptide is concerned, the results of the ten independent average conformations were all found to lie in the region of conformational space corresponding to the γ-turn. These results further support the conclusion that the γturn conformation is thermodynamically favored.     

Fast de novo discovery of low‐energy protein loop conformations

In the prediction of protein structure from amino acid sequence, loops are challenging regions for computational methods. Since loops are often located on the protein surface, they can have significant roles in determining protein functions and binding properties. Loop prediction without the aid of a structural template requires extensive conformational sampling and energy minimization, which are computationally difficult. In this article we present a new de novo loop sampling method, the Parallely filtered Energy Targeted All‐atom Loop Sampler (PETALS) to rapidly locate low energy conformations. PETALS explores both backbone and side‐chain positions of the loop region simultaneously according to the energy function selected by the user, and constructs a nonredundant ensemble of low energy loop conformations using filtering criteria. The method is illustrated with the DFIRE potential and DiSGro energy function for loops, and shown to be highly effective at discovering conformations with near‐native (or better) energy. Using the same energy function as the DiSGro algorithm, PETALS samples conformations with both lower RMSDs and lower energies. PETALS is also useful for assessing the accuracy of different energy functions. PETALS runs rapidly, requiring an average time cost of 10 minutes for a length 12 loop on a single 3.2 GHz processor core, comparable to the fastest existing de novo methods for generating an ensemble of conformations. Proteins 2017; 85:1402–1412. © 2017 Wiley Periodicals, Inc.     

A Monte Carlo method for generating structures of short single-stranded DNA sequences

A Monte Carlo method has been developed for generating the conformations of short single-stranded DNAs from arbitrary starting states. The chain conformers are constructed from energetically favorable arrangements of the constituent mononucleotides. Minimum energy states of individual dinucleotide monophosphate molecules are identified using a torsion angle minimizer. The glycosyl and acyclic backbone torsions of the dimers are allowed to vary, while the sugar rings are held fixed in one of the two preferred puckered forms. A total of 108 conformationally distinct states per dimer are considered in this first stage of minimization. The torsion angles within 5 kcal/mole of the global minimum in the resulting optimized states are then allowed to vary by ±10° in an effort to estimate the breadth of the different local minima. The energies of a total of 2187 (3⁷) angle combinations are examined per local conformational minimum. Finally, the energies of all dinucleotide conformers are scaled so that the populations of differently puckered sugar rings in the theoretical sample match those found in nmr solution studies. This last step is necessitated by limitations in the theoretical methods to predict DNA sugar puckering accurately. The conformer populations of the individual acyclic torsion angles in the composite dimer ensembles are found to be in good agreement with the distributions of backbone conformations deduced from nmr coupling constants and the frequencies of glycosyl conformations in x-ray crystal structures, suggesting that the low energy states are reasonable. The low energy dimer forms (consisting of 150–325 conformational states per dimer step) are next used as variables in a Monte Carlo algorithm, which generates the conformations of single-stranded d(CX_nG) chains, where X = A, T and n = 3, 4, 5. The oligonucleotides are built sequentially from the 5′ end of the chain using random numbers to select the conformations of overlapping dimer units. The simulations are very fast, involving a total of 10⁶ conformations per chain sequence. The potential errors in the buildup procedure are minimized by taking advantage of known rotational interdependences in the sugar–phosphate backbone. The distributions of oligonucleotide conformations are examined in terms of the magnitudes, positions, and orientations of the end-to-end vectors of the chains. The differences in overall flexibility and extension of the oligomers are discussed in terms of the conformations of the constituent dinucleotide steps, while the general methodology is discussed and compared with other nucleic acid model building techniques. © 1993 John Wiley & Sons, Inc.     

Cis-trans isomerization of proline in the peptide (his 105-Val 124) of ribonuclease a containing the primary nucleation site

Conformational energy calculations have been carried out to determine the relative stabilities of the C-terminal sequence 105–124 of ribonuclease A, withcis andtrans forms, respectively, of Asn 113-Pro 114. Thecis form of Pro 114 is the one that occurs in the native protein. This peptide contains the sequence 106–118, which, on the basis of both theoretical and experimental studies, is thought to constitute the primary nucleation site for the folding of ribonuclease A. It is shown that both conformations of the isolated peptide (with Pro 114 in thecis andtrans forms, respectively) are of approximately equal stability. Both forms have similar conformations from residues 105–110 and 118–124, while they differ in the bend region involving residues 111–117. Calculations have also been carried out to deduce the possible low-energy paths for the interconversion between thecis andtrans forms of both Pro 114 and Pro 117. It is shown that there are two low-energy paths (with a minimum activation energy of 16.5 kcal/mole) for the interconversion of Pro 114. Attractive nonbonded interaction energies stabilize the transition state on these paths. Only one relatively low-energy path (with an activation energy of 18 kcal/mole) could be found for the isomerization of Pro 117, which occur in thetrans form in the native protein; in this case, allcis forms have significantly higher energy than thetrans form. These calculations thus show that native-like forms for the isolated peptide can exist with Pro 114 in either thecis or thetrans form and that these forms are readily interconvertible.     

Experimental thermodynamics of the helix--random coil transition. 3. Determination of the transition enthalpies of the helical complexes poly(I+C) and poly I in solution

The enthalpies of the helix-coil transitions of the ordered polynucleotide systems of poly(inosinic acid)–poly(cytidylic acid) [poly(I + C)], (helical duplex), and of poly (inosinic acid) [poly(I + I + I)], (proposed secondary structure: a triple-stranded helical complex), were determined by using an adiabatic twin-vessel differential calorimeter. Measuring the temperature course of the heat capacity of the aqueous polymer solutions, the enthalpy values for the dissociation of the helical duplex poly (I + C) and the three-stranded helical complex poly(I + 1 + 1), respectively, were obtained by evaluating the additional heat capacity involved in the conformational change of the polynucleotide system in the transition range. The ΔH values of the helix-coil transition of poly (I + C) resulting from the analysis of the calorimetric measurements vary between the limits 6.5 ± 0.4 kcal/mole (I + C) and 8.4 ± 0.4 kcal/mole (I + C). depending on the variation of the cation concentration ranging from 0.063 mole cations kg H₂O to 1.003 mole cations/kg H₂O. The calorimetric investigation of an aqueous poly I solution (cation concentration 1.0 mole/kg H₂O) yielded the enthalpy value ΔH = 1.9 ± 0.4 kcal/mole (I), a result which has been interpreted qualitatively following current models of inter- and intramolecular forces of biologically significant macromolecules. Additional information on the transition behavior of poly(I+ C)Was obtained by ultraviolet and infrared absorption measurements.     

The helix-coil transitions of poly (dA)-poly (dT) and poly (dA-dT)-poly (dA-dT)

Helix–coil transition curves are calculated for poly (dA) poly(dT) and poly (dA-dT) poly (dA-dT) using the integral equation approach of Goel and Montroll.⁵ The transitions are described by the loop entropy model with the exponent of the loop entropy factor, k, remaining an arbitrary constant. The theoretical calculations are compared with experimental transition curves of the two polymers. Results indicate that the stacking energies for these two polymers differ by about 1 kcal/mole of base pairs. Also, a fit between theory and experiment was not possible for k > 1.70.     

Conformational analysis of chitobiose and chitosan

The relaxed potential energy surfaces of chitobiose were calculated based on the MM3-force field by optimizing dimer structures on a 10° grid spacing of the torsional angles about the glycosidic bonds (Φ,Ψ). The 36 conformations; the four combinations of the hydroxymethyl group orientations coupled with the nine of the secondary group ones— were assumed for each Φ,Ψ conformation. The four conformations, each differing in the hydroxymethyl group orientations, were considered for the whole Φ,Ψ space, and all the 36 conformations, for the restricted space of low energy. While the resulting energy map and the structures of the energy minima were similar to those proposed for cellobiose in many respects, more restricted energy profile was suggested for the relaxed map of chitobiose where differences in the energy level between the global minimum and the local minima were within 5.4 kcal/mol, compared with the equivalent value of 3.6 kcal/mol for cellobiose. Further depression of the global minimum occurred when the acidic residue was used. The Monte Carlo samples of the chitosan chain were generated based on the relaxed map to predict the unperturbed coil dimension in solution. The chitosan chains showed Gaussian behavior at x = 500 (x, degree of polymerization) and gave the characteristic ratio C_x, of about 70, which was much larger than the experimental values observed for the chitosan and cellulosic chains. © 1994 John Wiley & Sons, Inc.     

A molecular orbital study of the conformation of formycin

Semiempirical quantum mechanical calculations, using the iterative extended Huckel theory, are carried out for the evaluation of conformational energies, dipole moment and net atomic charges as a function of the rotation about the glycosidic bond. Torsion about the C(4′)-C(5′) bond has also been considered. The energy diagrams for either the gg or gt rotamers of formycin predict that neither the first or the second energy minimum fall in the classical anti or syn regions. The predicted energy difference between the two most preferred conformations is rather large (3 kcal/mole). In contrast adenosine is predicted to favor the anti conformation by less than 1 kcal/mole. Barriers to internal counter-clockwise rotation about the glycosidic bond are higher for adenosine.     

Variants of human thymidylate synthase with loop 181–197 stabilized in the inactive conformation

Loop 181–197 of human thymidylate synthase (hTS) populates two major conformations, essentially corresponding to the loop flipped by 180°. In one of the conformations, the catalytic Cys195 residue lies distant from the active site making the enzyme inactive. Ligands stabilizing this inactive conformation may function as allosteric inhibitors. To facilitate the search for such inhibitors, we have expressed and characterized several mutants designed to shift the equilibrium toward the inactive conformer. In most cases, the catalytic efficiency of the mutants was only somewhat impaired with values of k_cat/K_m reduced by factors in a 2–12 range. One of the mutants, M190K, is however unique in having the value of k_cat/K_m smaller by a factor of ～7500 than the wild type. The crystal structure of this mutant is similar to that of the wt hTS with loop 181–197 in the inactive conformation. However, the direct vicinity of the mutation, residues 188–194 of this loop, assumes a different conformation with the positions of C_α shifted up to 7.2 Å. This affects region 116–128, which became ordered in M190K while it is disordered in wt. The conformation of 116–128 is however different than that observed in hTS in the active conformation. The side chain of Lys190 does not form contacts and is in solvent region. The very low activity of M190K as compared to another mutant with a charged residue in this position, M190E, suggests that the protein is trapped in an inactive state that does not equilibrate easily with the active conformer.     

Conformation of the Ras-binding domain of Raf studied by molecular dynamics and free energy simulations

Recognition of Ras by its downstream target Raf is mediated by a Ras-recognition region in the Ras-binding domain (RBD) of Raf. Residues 78–89 in this region occupy two different conformations in the ensemble of NMR solution structures of the RBD: a fully α-helical one, and one where 87–90 form a type IV β-turn. Molecular dynamics simulations of the RBD in solution were performed to explore the stability of these and other possible conformations of both the wild-type RBD and the R89K mutant, which does not bind Ras. The simulations sample a fully helical conformation for residues 78–89 similar to the NMR helical structures, a conformation where 85–89 form a 3₁₀-helical turn, and a conformation where 87–90 form a type I |iB-turn, whose free energies are all within 0.3 kcal/mol of each other. NOE patterns and H^α chemical shifts from the simulations are in reasonable agreement with experiment. The NMR turn structure is calculated to be 3 kcal/mol higher than the three above conformations. In a simulation with the same implicit solvent model used in the NMR structure generation, the turn conformation relaxes into the fully helical conformation, illustrating possible structural artifacts introduced by the implicit solvent model. With the Raf R89K mutant, simulations sample a fully helical and a turn conformation, the turn being 0.9 kcal/mol more stable. Thus, the mutation affects the population of RBD conformations, and this is expected to affect Ras binding. For example, if the fully helical conformation of residues 78–89 is required for binding, its free energy increase in R89K will increase the binding free energy by about 0.6 kcal/mol. Proteins 31:186–200, 1998. © 1998 Wiley-Liss, Inc.     

Structural properties of amyloid β(1‐40) dimer explored by replica exchange molecular dynamics simulations

Replica exchange molecular dynamics simulations (300 ns) were used to study the dimerization of amyloid β(1‐40) (Aβ(1‐40)) polypeptide. Configurational entropy calculations revealed that at physiological temperature (310 K, 37°C) dynamic dimers are formed by randomly docked monomers. Free energy of binding of the two chains to each other was ?93.56 ± 6.341 kJ mol^?1. Prevalence of random coil conformations was found for both chains with the exceptions of increased β‐sheet content from residues 16‐21 and 29‐32 of chain A and residues 15‐21 and 30‐33 of chain B with β‐turn/β‐bend conformations in both chains from residues 1‐16, 21‐29 of chain A, 1‐16, and 21‐29 of chain B. There is a mixed β‐turn/β‐sheet region from residues 33‐38 of both chains. Analysis of intra‐ and interchain residue distances shows that, although the individual chains are highly flexible, the dimer system stays in a loosely packed antiparallel β‐sheet configuration with contacts between residues 17‐21 of chain A with residues 17‐21 and 31‐36 of chain B as well as residues 31‐36 of chain A with residues 17‐21 and 31‐36 of chain B. Based on dihedral principal component analysis, the antiparallel β‐sheet‐loop‐β‐sheet conformational motif is favored for many low energy sampled conformations. Our results show that Aβ(1‐40) can form dynamic dimers in aqueous solution that have significant conformational flexibility and are stabilized by collapse of the central and C‐terminal hydrophobic cores with the expected β‐sheet‐loop‐β‐sheet conformational motif. Proteins 2017; 85:1024–1045. © 2017 Wiley Periodicals, Inc.     

New developments of the electrostatically driven monte carlo method: Test on the membrane-bound portion of melittin

The electrostatically driven Monte Carlo (EDMC) method has been greatly improved by adding a series of new features, including a procedure for cluster analysis of the accepted conformations. This information is used to guide the search for the global energy minimum. Alternative procedures for generating perturbed conformations to sample the conformational space were also included. These procedures enhance the efficiency of the method by generating a larger number of low-energy conformations. The improved EDMC method has been used to explore the conformational space of a 20-residue polypeptide chain whose sequence corresponds to the membrane-bound portion of melittin. The ECEPP/3 (Empirical Conformational Energy Program for Peptides) algorithm was used to describe the conformational energy of the chain. After an exhaustive search involving 14 independent runs, the lowest energy conformation (LEC) (−91.0 kcal/mol) of the entire study was encountered in four of the runs, while conformations higher in energy by no more than 1.8 kcal/mol were found in the remaining runs with the exception of one of them (run 8). The LEC is identical to the conformation found recently by J. Lee, H.A. Scheraga, and S. Rackovsky [(1998) “Conformational Analysis of the 20-Residue Membrane-Bound Portion of Melittin by Conformational Space Annealing,” Biopolymers, Vol. 46, pp. 103–115] as the lowest energy conformation obtained in their study using the conformational space annealing method. These results suggest that this conformation corresponds to the global energy minimum of the ECEPP/3 potential function for this specific sequence; it also appears to be the conformation of lowest free energy. © 1998 John Wiley & Sons, Inc. Biopoly 46: 117–126, 1998     

Prediction of protein--ligand interactions: the complex of porcine pancreatic elastase with a valine-derived benzoxazinone

Prior to availability of the crystal structure of the complex, we evaluated models of the complex between porcine pancreatic elastase and a t-Boc–Val-derived benzoxazinone inhibitor. Models of the noncovalent and covalent complex were generated using computer graphics and each model was subjected to energy minimization using molecular mechanics. After the crystal structure became available, we found that the model with the lowest energy was in good agreement with the crystal structure, except for the position of the His57 side chain. Permissible conformations of the inhibitor were based on information from x-ray crystal structures and an earlier conformational energy investigation of t-Boc–amino acids. We did not, however, limit ourselves to these conformations. The conformation of the inhibitor in the lowest energy model and crystal structure, was not similar to any of the minimum-energy conformations of t-Boc–amino acids. This suggests that limiting proposed binding modes only to the lowest energy conformations of a ligand (prior to binding) may sometimes unfairly bias the procedure.     

Conformational energy calculations on glycosylated turns in glycoproteins

Analysis of the amino acid sequence of glycoproteins has suggested the β-turn as a likely site of glycosylation in glycoproteins. According to this model, the peptide chain traverses the interior of a globular protein, reversing its direction at the protein surface, a likely point for the attachment of hydrophilic carbohydrate residues. In order to search for plausible conformations of glycosylated β-turns in asparagine-linked glycoproteins, we have adapted the conformational energy calculation method of Scheraga and coworkers for use in carbohydrates. The parameters for nonbonded and hydrogen-bonded interactions have been published, and electrostatic parameters are derived from a CNDO calculation on a model glycopeptide. Our results indicate that the orientation of the glycosyl amide bond having the amide proton nearly trans to the anomeric proton of the sugar has the lowest energy. Although CD and nmr experiments in our laboratory have consistently found this conformation, our calculations show the conformation having these two protons in a cis relationship to lie very close in energy. Calculations on the glycopeptide linkage model, α-N-acetyl, δ-N(2-acetamido-1,2-dideoxy-β-D -glucopyranosyl)-N′-methyl-L -asparaginyl amide show that several distinct geometries are allowed for glycosylated β-turns. For a type I β-turn, three conformations of the glycosylated side chain are found within 4 kcal of the minimum, while two conformations of the glycosylated side chain are allowed for a type II turn. The hydrogen-bonded C7 conformation is also allowed. Stereoviews of the low-energy conformations reveal no major hydrogen-bonding interaction between the peptide and sugar.     

A facile synthesis of ceramides.

Lateral diffusion of phosphatide molecules in liquid crystalline bilayers has been analysed as a case of co-operative lattice diffusion. The potential energy of interaction between two molecules is assumed to arise from Van der Waals interactions of the hydrocarbon chains, and to have the form suggested by Salem [6]. From the observed values of the self-diffusion constant (of the order of 10^?8 cm² sec^?1) the depth of the potential “well” for two molecules at the equilibrium separation was estimated to have a lower limit of 1.95 kcal per mole, and the energy barrier to lateral motion was estimated to have an upper limit of 7.21 kcal per mole.     

Potentiometric estimation of charges in barnacle muscle fibers under internal perfusion

Summary A statistical mechanical treatment of the fluidity of lipid hydrocarbon chains in phospholipid bilayers is presented, which explicitly takes some account of interchain steric restrictions. With an effective energy separation of 750 cal/mole betweengauche andtrans conformations, it is found possible to account both for the chain dependence of the entropy and enthalpy change at the liquid crystalline transition of saturated lecithins, and also for intensity data in the laser raman spectra of dipalmitoyl lecithin. The method is used to calculate conformational probabilities in the lipid chains, in particular those for 2g 1 kinks. The calculated kink concentrations are found to be in agreement with the molecular permeability theory of H. Träuble (J Membrane Biol. 4:193, 1971).     

Role of connecting loop I in catalysis and allosteric regulation of human glucokinase

Glucokinase (GCK, hexokinase IV) is a monomeric enzyme with a single glucose binding site that displays steady‐state kinetic cooperativity, a functional characteristic that affords allosteric regulation of GCK activity. Structural evidence suggests that connecting loop I, comprised of residues 47–71, facilitates cooperativity by dictating the rate and scope of motions between the large and small domains of GCK. Here we investigate the impact of varying the length and amino acid sequence of connecting loop I upon GCK cooperativity. We find that sequential, single amino acid deletions from the C‐terminus of connecting loop I cause systematic decreases in cooperativity. Deleting up to two loop residues leaves the k_cat value unchanged; however, removing three or more residues reduces k_cat by 1000‐fold. In contrast, the glucose K_0.5 and K_D values are unaffected by shortening the connecting loop by up to six residues. Substituting alanine or glycine for proline‐66, which adopts a cis conformation in some GCK crystal structures, does not alter cooperativity, indicating that cis/trans isomerization of this loop residue does not govern slow conformational reorganizations linked to hysteresis. Replacing connecting loop I with the corresponding loop sequence from the catalytic domain of the noncooperative isozyme human hexokinase I (HK‐I) eliminates cooperativity without impacting the k_cat and glucose K_0.5 values. Our results indicate that catalytic turnover requires a minimal length of connecting loop I, whereas the loop has little impact upon the binding affinity of GCK for glucose. We propose a model in which the primary structure of connecting loop I affects cooperativity by influencing conformational dynamics, without altering the equilibrium distribution of GCK conformations.