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DOE OSTI2011

The Phe105 Loop of Alix Bro1 Domain Plays a Key Role in HIV-1 Release

Alix and cellular paralogs HD-PTP and Brox contain N-terminal Bro1 domains that bind ESCRT-III CHMP4. In contrast to HD-PTP and Brox, expression of the Bro1 domain of Alix alleviates HIV-1 release defects that result from interrupted access to ESCRT. In an attempt to elucidate this functional discrepancy, we solved the crystal structures of the Bro1 domains of HD-PTP and Brox. They revealed typical 'boomerang' folds they share with the Bro1 Alix domain. However, they each contain unique structural features that may be relevant to their specific function(s). In particular, phenylalanine residue in position 105 (Phe105) of Alix belongs to a long loop that is unique to its Bro1 domain. Concurrently, mutation of Phe105 and surrounding residues at the tip of the loop compromise the function of Alix in HIV-1 budding without affecting its interactions with Gag or CHMP4. These studies identify a new functional determinant in the Bro1 domain of Alix.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2015

Computational Study of Symmetric Methylation on Histone Arginine Catalyzed by Protein Arginine Methyltransferase PRMT5 through QM/MM MD and Free Energy Simulations

Protein arginine methyltransferases (PRMTs) catalyze the transfer of the methyl group from S-adenosyl-l-methionine (AdoMet) to arginine residues. There are three types of PRMTs (I, II and III) that produce different methylation products, including asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and monomethylarginine (MMA). Since these different methylations can lead to different biological consequences, understanding the origin of product specificity of PRMTs is of considerable interest. In this article, the quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy simulations are performed to study SDMA catalyzed by the Type II PRMT5 on the basis of experimental observation that the dimethylated product is generated through a distributive fashion. The simulations have identified some important interactions and proton transfers during the catalysis. Similar to the cases involving Type I PRMTs, a conserved Glu residue (Glu435) in PRMT5 is suggested to function as general base catalyst based on the result of the simulations. Moreover, our results show that PRMT5 has an energetic preference for the first methylation on N-1 followed by the second methylation on a different -guanidino nitrogen of arginine (N-2).The first and second methyl transfers are estimated to have free energy barriers of 19-20 and 18-19 kcal/mol respectively. The computer simulations suggest a distinctive catalytic mechanism of symmetric dimethylation that seems to be different from asymmetric dimethylation.

ARABIDOPSIS↗

DOE OSTI2016

Structural basis for germline antibody recognition of HIV-1 immunogens

Efforts to elicit broadly neutralizing antibodies (bNAbs) against HIV-1 require understanding germline bNAb recognition of HIV-1 envelope glycoprotein (Env). The VRC01-class bNAb family derived from the VH1-2*02 germline allele arose in multiple HIV-1–infected donors, yet targets the CD4-binding site on Env with common interactions. Modified forms of the 426c Env that activate germline-reverted B cell receptors are candidate immunogens for eliciting VRC01-class bNAbs. We present structures of germline-reverted VRC01-class bNAbs alone and complexed with 426c-based gp120 immunogens. Germline bNAb–426c gp120 complexes showed preservation of VRC01-class signature residues and gp120 contacts, but detectably different binding modes compared to mature bNAb-gp120 complexes. Unlike typical antibody-antigen interactions, VRC01–class germline antibodies exhibited preformed antigen-binding conformations for recognizing immunogens. Affinity maturation introduced substitutions increasing induced-fit recognition and electropositivity, potentially to accommodate negatively-charged complex-type N-glycans on gp120. These results provide general principles relevant to the unusual evolution of VRC01–class bNAbs and guidelines for structure-based immunogen design.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

Development and characterization of an ice-selecting pumped counterflow virtual impactor (IS-PCVI) to study ice crystal residuals

Separation of particles that play a role in cloud activation and ice nucleation from interstitial aerosols has become necessary to further understand aerosol-cloud interactions. The pumped counterflow virtual impactor (PCVI), which uses a vacuum pump to accelerate the particles and increase their momentum, provides an accessible option for dynamic and inertial separation of cloud elements. However, the use of a traditional PCVI to extract large cloud hydrometeors is difficult mainly due to its small cut-size diameters (< 5 µm). Here, for the first time we describe a development of an ice-selecting PCVI (IS-PCVI) to separate ice in controlled mixed-phase cloud system based on the particle inertia with the cut-off diameter ≥ 10 µm. We also present its laboratory application demonstrating the use of the impactor under a wide range of temperature and humidity conditions. The computational fluid dynamics simulations were initially carried out to guide the design of the IS-PCVI. After fabrication, a series of validation laboratory experiments were performed coupled with the Aerosol Interaction and Dynamics in the Atmosphere (AIDA) expansion cloud simulation chamber. In the AIDA chamber, test aerosol particles were exposed to the ice supersaturation conditions (i.e., RH ice > 100 %), where a mixture of droplets and ice crystals was formed during the expansion experiment. In parallel, the flow conditions of the IS-PCVI were actively controlled, such that it separated ice crystals from a mixture of ice crystals and cloud droplets, which were of diameter ≥ 10 µm. These large ice crystals were passed through the heated evaporation section to remove the water content. Afterwards, the residuals were characterized with a suite of online and offline instruments downstream of the IS-PCVI. These results were used to assess the optimized operating parameters of the device in terms of (1) the critical cut-size diameter, (2) the transmission efficiency and (3) the counterflow-to-input flow ratio. Particle losses were characterized by comparing the residual number concentration to the rejected interstitial particle number concentration. Overall results suggest that the IS-PCVI enables inertial separation of particles with a volume-equivalent particle size in the range of ~ 10–30 µm in diameter with small inadvertent intrusion (~  5 %) of unwanted particles.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2023

Chromophore carbonyl twisting in fluorescent biosensors encodes direct readout of protein conformations with multicolor switching

Fluorescent labeling of proteins is a powerful tool for probing structure-function relationships with many biosensing applications. Structure-based rules for systematically designing fluorescent biosensors require understanding ligand-mediated fluorescent response mechanisms which can be challenging to establish. We installed thiol-reactive derivatives of the naphthalene-based fluorophore Prodan into bacterial periplasmic glucose-binding proteins. Glucose binding elicited paired color exchanges in the excited and ground states of these conjugates. X-ray structures and mutagenesis studies established that glucose-mediated color switching arises from steric interactions that couple protein conformational changes to twisting of the Prodan carbonyl relative to its naphthalene plane. Mutations of residues contacting the carbonyl can optimize color switching by altering fluorophore conformational equilibria in the apo and glucose-bound proteins. A commonly accepted view is that Prodan derivatives report on protein conformations via solvatochromic effects due to changes in the dielectric of their local environment. Here we show that instead Prodan carbonyl twisting controls color switching. These insights enable structure-based biosensor design by coupling ligand-mediated protein conformational changes to internal chromophore twists through specific steric interactions between fluorophore and protein.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

NASA NTRS1977

Ancient carbon and noble gas fractionation

Noble gases in ancient terrestrial kerogen and meteoritic carbonaceous residues are compared in terms of their elemental fractionation with respect to atmospheric and the cosmic reservoirs, respectively. Fractionation factors for the heavy noble gases are almost identical in both types of samples. Therefore, some features of the interaction between carbon phases and noble gases in very different environments of origin appear to be similar. These findings underscore the plausibility of the meteoritic carbonaceous residues as a noble gas carrier and as a novel vehicle for achieving the elemental fractionation required to derive the planetary noble gas pattern from cosmic abundances.

Frick, U.↗

DOE OSTI2015

Secondary PDZ domain-binding site on class B plexins enhances the affinity for PDZ–RhoGEF

PDZ domains are abundant protein interaction modules and typically recognize a short motif at the C terminus of their ligands, with a few residues in the motif endowing the binding specificity. The sequence-based rules, however, cannot fully account for the specificity between the vast number of PDZ domains and ligands in the cell. Plexins are transmembrane receptors that regulate processes such as axon guidance and angiogenesis. Two related guanine nucleotide exchange factors (GEFs), PDZ–RhoGEF and leukemia-associated RhoGEF (LARG), use their PDZ domains to bind class B plexins and play critical roles in signaling. Here, we present the crystal structure of the full-length cytoplasmic region of PlexinB2 in complex with the PDZ domain of PDZ–RhoGEF. The structure reveals that, in addition to the canonical C-terminal motif/PDZ interaction, the 3D domain of PlexinB2 forms a secondary interface with the PDZ domain. Our biophysical and cell-based assays show that the secondary interface contributes to the specific interaction between plexin and PDZ–RhoGEF and to signaling by plexin in the cell. As a result, formation of secondary interfaces may be a general mechanism for increasing affinity and specificity of modular domain-mediated interactions.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Incorporating Lanthanum into Mesoporous Silica Foam Enhances Enzyme Immobilization and the Activity of Penicillin G Acylase Due to Lewis Acid-Base Interactions

Penicillin G acylase (PGA) has been immobilized on a lanthanum-incorporated mesostructured cellular foam (La-MCF) support by using the interaction between the strong Lewis acid sites on the surface of La-MCF and the free amino groups of lysine residues of PGA. The La-MCF support was successfully synthesized in situ through the addition of a citric acid (CA) complexant. The results of pyridine-IR spectroscopy show the presence of strong Lewis acid sites on the surface of the prepared La-MCF (with CA), attributed to the incorporation of lanthanum species into the framework of MCF. Through interaction with the strong Lewis acid sites, the enzymes can be firmly immobilized on the surface of the support. The results indicate that PGA/La-MCF (with CA) exhibits a high specific activity and greatly enhanced operational stability. For the hydrolysis of penicillin G potassium salt, the initial specific activity of PGA/La-MCF (with CA) reaches 10023 U/g. Finally, even after being recycled 10 times, PGA/La-MCF (with CA) retains 89 % of its initial specific activity, much higher than the 77 % of PGA/Si-MCF.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2011

Structure and Function of APH(4)-Ia, a Hygromycin B Resistance Enzyme

The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 {angstrom} resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity, indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2{double_prime}) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme.

08 HYDROGEN↗

DOE OSTI2017

Enhancement of TGF-β-induced Smad3 activity by c-Abl-mediated tyrosine phosphorylation of its coactivator SKI-interacting protein (SKIP)

c-Abl is a non-receptor-type tyrosine kinase that plays an important role in cell proliferation, migration, apoptosis, and fibrosis. Furthermore, although c-Abl is involved in transforming growth factor-β (TGF-β) signaling, its molecular functions in TGF-β signaling are not fully understood. Here, we found that c-Abl phosphorylates SKI-interacting protein (SKIP), a nuclear cofactor of the transcription factor Smad3. The c-Abl inhibitor imatinib suppressed TGF-β-induced expression of Smad3 targets as well as SKIP/Smad3 interaction. TGF-β-stimulation induced tyrosine phosphorylation of SKIP, and this phosphorylation was suppressed by imatinib. Tyr{sup 292}, Tyr{sup 430}, and Tyr{sup 433} residues in SKIP were shown to be involved in c-Abl-mediated phosphorylation. Phosphomimetic glutamic acid substitution at Tyr{sup 292} in SKIP enhanced, whereas its phospho-dead phenylalanine substitution attenuated TGF-β-induced SKIP/Smad3 interaction. Moreover, the phosphomimetic mutant of SKIP augmented transcriptional activity of Smad3. Taken together, these results suggest that c-Abl phosphorylates SKIP mainly at Tyr{sup 292} and promotes SKIP/Smad3 interaction for the full activation of TGF-β/Smad3 signaling. - Highlights: • c-Abl promotes TGF-β-induced SKIP/Smad3 interaction. • c-Abl phosphorylates SKIP upon TGF-β stimulation. • Phosphorylation of SKIP at Tyr{sup 292} enhances SKIP/Smad3 binding and Smad3 activity.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2014

Complete particle-pair annihilation as a dynamical signature of the spectral singularity

Motivated by the physical relevance of a spectral singularity of interacting many-particle system, we explore the dynamics of two bosons as well as fermions in one-dimensional system with imaginary delta interaction strength. Based on the exact solution, it shows that the two-particle collision leads to amplitude-reduction of the wave function. For fermion pair, the amplitude-reduction depends on the spin configuration of two particles. In both cases, the residual amplitude can vanish when the relative group velocity of two single-particle Gaussian wave packets with equal width reaches the magnitude of the interaction strength, exhibiting complete particle-pair annihilation at the spectral singularity. - Highlights: • We investigate the physical relevance of a spectral singularity. • The two-particle collision leads to amplitude-reduction of the wave function. • There is a singularity spectrum which leads to complete particle-pair annihilation. • Complete particle-pair annihilation can only occur for two distinguishable bosons and singlet fermions. • Pair annihilation provides a detection method of the spectral singularity in the experiment.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗

DOE OSTI2012

Structures of the lamin A/C R335W and E347K mutants: Implications for dilated cardiolaminopathies

Highlights: Black-Right-Pointing-Pointer Defects in the Lamin AC gene have been linked to more than 10 laminopathies. Black-Right-Pointing-Pointer R335 and E347 are highly conserved residues, whose mutation preserves Coil2B 3D structure. Black-Right-Pointing-Pointer R335W and E347K mutations may affect LMNA interaction with proteins of nuclear lamina. -- Abstract: Dilated cardiomyopathy (DCM) is a condition whereby the normal muscular function of the myocardium is altered by specific or multiple aetiologies. About 25-35% of DCM patients show familial forms of the disease, with most mutations affecting genes encoding cytoskeletal proteins. Most of the DCM-related mutations fall in the Lamin AC gene, in particular in the Coil2B domain of the encoded protein. In this context, we focussed our studies on the crystal structures of two lamin Coil2B domain mutants (R335W and E347K). Both R335 and E347 are higly conserved residues whose substitution has little effects on the Coil2B domain three-dimensional structure; we can thus hypothesize that the mutations may interfere with the binding of components within the nuclear lamina, or of nuclear factors, that have been proposed to interact/associate with lamin A/C.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2024

CryoEM structures of the human CLC-2 voltage-gated chloride channel reveal a ball-and-chain gating mechanism

CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different tissues. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating among closely related homologs has been a long-standing mystery, in part because few CLC channel structures are available. Here, we report cryoEM structures of human CLC-2 at 2.46 – 2.76 Å, in the presence and absence of the selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl – -permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct conformations involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl – -permeation pathway. This peptide is highly conserved among species variants of CLC-2 but is not present in other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a “ball-and-chain” gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl – -permeation pathway.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2012

Structural Basis of Substrate Recognition in Human Nicotinamide N-Methyltransferase

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide, pyridines, and other analogues using S-adenosyl-L-methionine as donor. NNMT plays a significant role in the regulation of metabolic pathways and is expressed at markedly high levels in several kinds of cancers, presenting it as a potential molecular target for cancer therapy. We have determined the crystal structure of human NNMT as a ternary complex bound to both the demethylated donor S-adenosyl-L-homocysteine and the acceptor substrate nicotinamide, to 2.7 {angstrom} resolution. These studies reveal the structural basis for nicotinamide binding and highlight several residues in the active site which may play roles in nicotinamide recognition and NNMT catalysis. The functional importance of these residues was probed by mutagenesis. Of three residues near the nicotinamide's amide group, substitution of S201 and S213 had no effect on enzyme activity while replacement of D197 dramatically decreased activity. Substitutions of Y20, whose side chain hydroxyl interacts with both the nicotinamide aromatic ring and AdoHcy carboxylate, also compromised activity. Enzyme kinetics analysis revealed k{sub cat}/K{sub m} decreases of 2-3 orders of magnitude for the D197A and Y20A mutants, confirming the functional importance of these active site residues. The mutants exhibited substantially increased K{sub m} for both NCA and AdoMet and modestly decreased k{sub cat}. MD simulations revealed long-range conformational effects which provide an explanation for the large increase in K{sub m}(AdoMet) for the D197A mutant, which interacts directly only with nicotinamide in the ternary complex crystal structure.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

The Energetic Origins of Pi–Pi Contacts in Proteins

Accurate potential energy models of proteins must describe the many different types of noncovalent interactions that contribute to a protein’s stability and structure. Pi–pi contacts are ubiquitous structural motifs in all proteins, occurring between aromatic and nonaromatic residues and play a nontrivial role in protein folding and in the formation of biomolecular condensates. Guided by a geometric criterion for isolating pi–pi contacts from classical molecular dynamics simulations of proteins, we use quantum mechanical energy decomposition analysis to determine the molecular interactions that stabilize different pi–pi contact motifs. We find that neutral pi–pi interactions in proteins are dominated by Pauli repulsion and London dispersion rather than repulsive quadrupole electrostatics, which is central to the textbook Hunter–Sanders model. This results in a notable lack of variability in the interaction profiles of neutral pi–pi contacts even with extreme changes in the dielectric medium, explaining the prevalence of pi-stacked arrangements in and between proteins. We also find interactions involving pi-containing anions and cations to be extremely malleable, interacting like neutral pi–pi contacts in polar media and like typical ion–pi interactions in nonpolar environments. Like-charged pairs such as arginine–arginine contacts are particularly sensitive to the polarity of their immediate surroundings and exhibit canonical pi–pi stacking behavior only if the interaction is mediated by environmental effects, such as aqueous solvation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2015

Molecular design for growth of supramolecular membranes with hierarchical structure

Membranes with hierarchical structure exist in biological systems, and bio-inspired building blocks have been used to grow synthetic analogues in the laboratory through self-assembly. The formation of these synthetic membranes is initiated at the interface of two aqueous solutions, one containing cationic peptide amphiphiles (PA) and the other containing the anionic biopolymer hyaluronic acid (HA). The membrane growth process starts within milliseconds of interface formation and continues over much longer timescales to generate robust membranes with supramolecular PA–HA nanofibers oriented orthogonal to the interface. Computer simulation indicates that formation of these hierarchically structured membranes requires strong interactions between molecular components at early time points in order to generate a diffusion barrier between both solutions. Experimental studies using structurally designed PAs confirm simulation results by showing that only PAs with high ζ potential are able to yield hierarchically structured membranes. Furthermore, the chemical structure of such PAs must incorporate residues that form β-sheets, which facilitates self-assembly of long nanofibers. In contrast, PAs that form low aspect ratio nanostructures interact weakly with HA and yield membranes that exhibit non-fibrous fingering protrusions. Furthermore, experimental results show that increasing HA molecular weight decreases the growth rate of orthogonal nanofibers. Overall, this result is supported by simulation results suggesting that the thickness of the interfacial contact layer generated immediately after initiation of self-assembly increases with polymer molecular weight.

36 MATERIALS SCIENCE↗

DOE OSTI2011

Structural investigation of inhibitor designs targeting 3-dehydroquinate dehydratase from the shikimate pathway of Mycobacterium tuberculosis

The shikimate pathway is essential in Mycobacterium tuberculosis and its absence from humans makes the enzymes of this pathway potential drug targets. In the present paper, we provide structural insights into ligand and inhibitor binding to 3-dehydroquinate dehydratase (dehydroquinase) from M. tuberculosis (MtDHQase), the third enzyme of the shikimate pathway. The enzyme has been crystallized in complex with its reaction product, 3-dehydroshikimate, and with six different competitive inhibitors. The inhibitor 2,3-anhydroquinate mimics the flattened enol/enolate reaction intermediate and serves as an anchor molecule for four of the inhibitors investigated. MtDHQase also forms a complex with citrazinic acid, a planar analogue of the reaction product. The structure of MtDHQase in complex with a 2,3-anhydroquinate moiety attached to a biaryl group shows that this group extends to an active-site subpocket inducing significant structural rearrangement. The flexible extensions of inhibitors designed to form {pi}-stacking interactions with the catalytic Tyr{sup 24} have been investigated. The high-resolution crystal structures of the MtDHQase complexes provide structural evidence for the role of the loop residues 19-24 in MtDHQase ligand binding and catalytic mechanism and provide a rationale for the design and efficacy of inhibitors.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

Evaluating the catalytic importance of a conserved Glu97 residue in triosephosphate isomerase

Investigating enzyme activity is central to our understanding of biological function, and the design of biocatalysts continues to find applications in synthesis. While a role for active site residues can be proposed based on structure and mechanism, our understanding of the catalytic importance for residues surrounding the active site is less well understood. In triosephosphate isomerase (TIM), Glu97 is situated adjacent to the active site and is found in essentially all sequences. Prior studies reported mutation of Glu97 to Asp and Gln in TIM from Plasmodium falciparum (PfTIM) led to a 100- and 4000-fold decrease in activity, respectively, while the E97D mutation in TIM from Gallus gallus (cTIM) had no effect on activity. To investigate further the question of how mutations in essentially superimposable structures give different effects, we mutated E97 in TIM from Trypanosoma brucei brucei (TbbTIM), Saccharomyces cerevisiae (yTIM), and human (hTIM). The E97D, E97A, and E97Q mutations led to a ∼three-tenfold decrease in activity, a modest effect compared to the 10{sup 2}–10{sup 3}-fold effect in PfTIM. CD and fluorescence studies showed the overall structures for the mutants were essentially unchanged. Structural analysis shows that several residues surrounding E97 differ between PfTIM and TIM from the other organisms, and rearrangements or mispositioning of residues in PfTIM may lead to the different rate effects. The results illustrate the interplay of active site and surrounding residues in affecting catalysis and highlight that understanding of the role of residues surrounding the active site may aid in the incorporation of favorable or avoidance of unfavorable interactions when designing enzymes.

60 APPLIED LIFE SCIENCES↗