Dipeptide interactions with Zn(II)–cyclen artificial model for molecular recognition

The Zn(II)–cyclen–dipeptide ternary systems (where cyclen is abbreviated as L and dipeptide is glycylglycine (HL¹) or glycyl‐(S)‐alanine (HL²)) were investigated by potentiometry applying both “out‐of‐cell” and direct titrations and by ¹H NMR spectroscopy. Especially, the ¹H NMR study was found to be very efficient to estimate speciation in the systems. The results obtained under full equilibria indicated two main species, [Zn(L)(HL^1,2)]²⁺ and [Zn(L)(L^1,2)]⁺, in both the systems. In the [Zn(L)(HL^1,2)]²⁺ complex, presence of carbonyl‐carboxylate chelate was confirmed, and in the [Zn(L)(L^1,2)]⁺ species, the peptide coordination is re‐organized to carbonyl‐amine chelate or only terminal amino group is coordinated. Equilibrium constants describing [Zn(L)]²⁺–dipeptide interaction are relatively low, log K = 3.4 for Gly‐Gly and 4.1 for Gly‐(S)‐Ala, respectively. Nevertheless, the values are slightly higher than stability constants for interaction of Zn(II) with the dipeptides (i.e. [Zn(L^1,2)]⁺ species) where a chelate formation is expected. It indicates that interaction between Zn(II) ion in [Zn(L)]²⁺ and the dipeptides should be supported by some additional interactions. Potentiometry carried out under non‐equilibrum condition showed different species where these additional stabilizing forces play more important role. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterisation of H+ and Cu2+ binding to humic and fulvic acids

Abstract

The nature of H⁺ and Cu²⁺ binding by soil-derived humic (HA) and fulvic (FA) acid was characterised using potentiometric titrations. The experimental data obtained showed that the derived proton balance equation was valid and capable of describing proton consumption by both polyelectrolytes. HA was found to be more acidic and more reactive as shown by its lower equivalent weight compared to FA. Acid consumption by HA during titrations was little affected at ionic strength (μ) up to 0.1 M although it was enhanced at higher μ. Displacement of protons by Cu²⁺ resulted in a nonlinear sigmoidal pattern suggesting the formation of different Cu-HA chelates, or existence of sites that differed in their affinities for Cu on the ligand. Different concentrations of added Cu appeared to favour one or both mechanisms, although the titration method could not differentiate which of the probable mechanisms was more dominant at a specific level of Cu added. Similar values were obtained for conditional stability constants using either the equation of Scatchard or Ruzic.     

An improved synthesis of (2S, 4S)‐ and (2S, 4R)‐2‐amino‐4‐methyldecanoic acids: assignment of the stereochemistry of culicinins

An improved synthesis of (2S, 4S)‐ and (2S, 4R)‐2‐amino‐4‐methyldecanoic acids was accomplished using a glutamate derivative as starting material and Evans' asymmetric alkylation as the decisive step. The NMR data of the two diastereomers were measured and compared with those of the natural product. As a result, the stereochemistry of this novel amino acid unit in culicinins was assigned as (2S, 4R). Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

4‐Fluorophenyl 3‐nitro‐2‐pyridinesulfenate as a practical protecting agent for amino acids

We report a new protecting agent ( 1 , Npys‐OPh(pF)) for 3‐nitro‐2‐pyridine (Npy) sulfenylation of amino, hydroxy, and thiol functional groups. Several Npys phenoxides were synthesized from Npys chloride (Npys‐Cl) and phenols in the presence of base in 1‐step reaction, and their ability for Npy‐sulfenylation was evaluated. As a result, 1 was selected as a new Npy‐sulfenylation agent with advantages including improved physicochemical stability, more controllable reactivity, and easier handling than the conventional protecting agent Npys‐Cl.     

TAPP analogs containing β3‐homo‐amino acids: synthesis and receptor binding

β‐Amino acids containing α,β‐hybrid peptides show great potential as peptidomimetics. In this paper, we describe the synthesis and affinity to μ‐opioid and δ‐opioid receptors of α,β‐hybrids, analogs of the tetrapeptide Tyr‐ d ‐Ala‐Phe‐Phe‐NH₂ (TAPP). Each amino acid was replaced with an l ‐ or d ‐β³‐h‐amino acid. All α,β‐hybrids of TAPP analogs were synthesized in solution and tested for affinity to μ‐opioid and δ‐opioid receptors. The analog Tyr‐β³h‐ d ‐Ala‐Phe‐PheNH₂ was found to be as active as the native tetrapeptide. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and receptor binding of opioid peptide analogues containing β3‐homo‐amino acids

β‐Amino acids containing hybrid peptides and β‐peptides show great potential as peptidomimetics. In this paper we describe the synthesis and affinity toward the µ‐ and δ‐opioid receptors of β‐peptides, analogues of Leu‐enkephalin, deltorphin I, dermorphin and α,β‐hybrides, analogues of deltorphin I. Substitution of α‐amino acid residues with β³‐homo‐amino acid residues, in general resulted in decrease of affinity to opioid receptors. However, the incorporation β³h‐D ‐Ala in position 2 or β³hPhe in position 3 of deltorphin I resulted in potent and selective ligand for δ‐opioid receptor. The NMR studies of β‐deltorphin I analogue suggest that conformational motions in the central part of the peptide backbone are partially restricted and some conformational preferences can be expected. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Partitioning of amino acids in the aqueous biphasic system containing the water‐miscible ionic liquid 1‐butyl‐3‐methylimidazolium bromide and the water‐structuring salt potassium citrate

In biotechnology, extraction by means of aqueous biphasic systems (ABS) is known as a promising tool for the recovery and purification of bio‐molecules. Over the past decade, the increasing emphasis on cleaner and environmentally benign extraction procedures has led to enhanced interest in the ABS containing ionic liquids (ILs)—a new class of non‐volatile alternative solvents. ABS composed of the hydrophilic IL {1‐butyl‐3‐methylimidazolium bromide ([C₄mim]Br)} and potassium citrate—which is easily degraded—represents a clean media to green separation of bio‐molecules. In this regard, here, the extraction capability of this ABS was evaluated through its application to the extraction of some amino acids. To gain an insight into the driving forces of amino acid partitioning in the studied IL ‐based ABS, the distribution of five model amino acids (L ‐tryptophan, L ‐phenylalanine, L ‐tyrosine, L ‐leucine, and L ‐valine) at different aqueous medium pH values and different phase compositions was investigated. The studies indicated that hydrophobic interactions were the main driving force, although electrostatic interactions and salting‐out effects were also important for the transfer of the amino acids. Moreover, based on the statistical analysis of the driving forces of amino acid partitioning in the studied IL ‐based ABS, a model was established to describe the partition coefficient of three model amino acids, L ‐tryptophan, L ‐phenylalanine, and L ‐valine, and employed to predict the partition coefficient of two other model amino acids, L ‐tyrosine and L ‐leucine. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Acid–base and metal ion binding properties of 2-thiocytidine in aqueous solution

The thionucleoside 2-thiocytidine (C2S) occurs in nature in transfer RNAs; it receives attention in diverse fields like drug research and nanotechnology. By potentiometric pH titrations we measured the acidity constants of H(C2S)(+) and the stability constants of the M(C2S)(2+) and M(C2S-H)(+) complexes (M(2+) = Zn(2+), Cd(2+)), and we compared these results with those obtained previously for its parent nucleoside, cytidine (Cyd). Replacement of the (C2)=O unit by (C2)=S facilitates the release of the proton from (N3)H(+) in H(C2S)(+) (pK (a) = 3.44) somewhat, compared with H(Cyd)(+) (pK (a) = 4.24). This moderate effect of about 0.8 pK units contrasts with the strong acidification of about 4 pK units of the (C4)NH(2) group in C2S (pK (a) = 12.65) compared with Cyd (pK (a) approximately 16.7); the reason for this result is that the amino-thione tautomer, which dominates for the neutral C2S molecule, is transformed upon deprotonation into the imino-thioate form with the negative charge largely located on the sulfur. In the M(C2S)(2+) complexes the (C2)S group is the primary binding site rather than N3 as is the case in the M(Cyd)(2+) complexes, though owing to chelate formation N3 is to some extent still involved in metal ion binding. Similarly, in the Zn(C2S-H)(+) and Cd(C2S-H)(+) complexes the main metal ion binding site is the (C2)S(-) unit (formation degree above 99.99% compared with that of N3). However, again a large degree of chelate formation with N3 must be surmised for the M(C2S-H)(+) species in accord with previous solid-state studies of related ligands. Upon metal ion binding, the deprotonation of the (C4)NH(2) group (pK (a) = 12.65) is dramatically acidified (pK (a) approximately 3), confirming the very high stability of the M(C2S-H)(+) complexes. To conclude, the hydrogen-bonding and metal ion complex forming capabilities of C2S differ strongly from those of its parent Cyd; this must have consequences for the properties of those RNAs which contain this thionucleoside.     

Probing the helical forms of Ca2+‐guluronan junction zones in alginate gels by molecular dynamics 1: Duplexes

A molecular dynamics investigation of the helical forms adopted by (1→4)‐α‐L ‐guluronan in explicit water environment was carried out. Single chains and duplexes were modeled at 300 K starting both from 2₁ or 3₂ helical conformations and in the presence of a neutralizing amount of Ca²⁺ ions. All systems were allowed full conformational freedom. The initial perfect helices with integral screw symmetries were lost at the very beginning of simulations and two distinct behaviors were observed: At equilibrium the 2₁ models mostly retained the 2₁ local helical conformations while exploring the 3₂ ones the rest of the time. In duplexes the two chains, which behaved similarly, were well extended and slightly twisted. By contrast, the chains in 3₂ duplex models were dissimilar and explored a much broader conformational space in which 2₁ and 3₂ local helical conformations were dominant and equally represented but the 3₁ and other conformations were also present. The wide variety of conformations revealed in this study is consistent with the general difficulty in obtaining crystals of Ca²⁺‐guluronate with suitable lateral dimensions for crystallographic studies. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 562–571, 2013.     

Analysis of Ca2+/Mg2+ selectivity in α‐lactalbumin and Ca2+‐binding lysozyme reveals a distinct Mg2+‐specific site in lysozyme

The triggering of Ca²⁺ signaling pathways relies on Ca²⁺/Mg²⁺ specificity of proteins mediating these pathways. Two homologous milk Ca²⁺‐binding proteins, bovine α‐lactalbumin (bLA) and equine lysozyme (EQL), were analyzed using the simplest “four‐state” scheme of metal‐ and temperature‐induced structural changes in a protein. The association of Ca²⁺/Mg²⁺ by native proteins is entropy‐driven. Both proteins exhibit strong temperature dependences of apparent affinities to Ca²⁺ and Mg²⁺, due to low thermal stabilities of their apo‐forms and relatively high unfavorable enthalpies of Mg²⁺ association. The ratios of their apparent affinities to Ca²⁺ and Mg²⁺, being unusually high at low temperatures (5.3–6.5 orders of magnitude), reach the values inherent to classical EF‐hand motifs at physiological temperatures. The comparison of phase diagrams predicted within the model of competitive Ca²⁺ and Mg²⁺ binding with experimental data strongly suggests that the association of Ca²⁺ and Mg²⁺ ions with bLA is a competitive process, whereas the primary Mg²⁺ site of EQL is different from its Ca²⁺‐binding site. The later conclusion is corroborated by qualitatively different molar ellipticity changes in near‐UV region accompanying Mg²⁺ and Ca²⁺ association. The Ca²⁺/Mg²⁺ selectivity of Mg²⁺‐site of EQL is below an order of magnitude. EQL exhibits a distinct Mg²⁺‐specific site, probably arising as an adaptation to the extracellular environment. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Chiral Recognition of N‐Phthaloyl,N‐Tetrachlorophthaloyl,and N‐Naphthaloyl α‐Amino Acids and Their Esters on Polysaccharide‐Derived Chiral Stationary Phases

Enantiomeric separations of N‐phthaloyl (N‐PHT), N‐tetrachlorophthaloyl (N‐TCPHT), and N‐naphthaloyl (N‐NPHT) α‐amino acids and their esters were examined on several kinds of polysaccharide‐derived chiral stationary phases (CSPs). Resolution capability of CSPs was greater Chiralcel OF than the others for N‐PHT and N‐NPHT α‐amino acids and their esters. In N‐TCPHT α‐amino acids and their esters, good enantioselectivities showed Chiralcel OG for N‐TCPHT α‐amino acids, Chiralpak AD for N‐TCPHT α‐amino acid methyl esters, and Chiralcel OD for N‐TCPHT α‐amino acid ethyl esters, respectively. From the results of liquid chromatography and computational chemistry, it is concluded that l ‐form is preferred and more retained with electrostatic interaction in case of interaction between N‐PHT α‐amino acid derivatives and Chiralcel OF, N‐TCPHT α‐amino acid derivatives and Chiralcel OD, and N‐NPHT α‐amino acid derivatives and Chiracel OF. On the other hand, d ‐form is preferred and more retained with van der Waals interaction in case of interaction between N‐TCPHT α‐amino acid ester derivatives and Chiralcel OG and Chiralpak AD. Chirality 24:1037–1046, 2012. © 2012 Wiley Periodicals, Inc.     

A comparison of convergently evolved insect silks that share β‐sheet molecular structure

Raspy crickets produce silk webs that are used to build shelters. These webs have been found to consist of both fiber and film components. Raman spectra obtained from both components were found to be very similar for a given species. The protein structure of the fibers and films produced by both species was predominately β‐sheet with lesser amounts of β‐turns, unordered and α‐helical protein also detected. The orientation of the β‐sheet backbone in the fiber was determined to be parallel to the fiber axis. Compared to cocoon and dragline silk the orientation distribution exhibits a significant randomly orientated protein component. Amino acid analysis confirmed the presence of glycine, serine, and alanine in both species, which are known to form antiparallel β‐sheet structures. Both species, although at significantly different concentrations, where found to contain proline. This amino acid is uncommon in insect silks, and likely involved in increasing fiber elasticity. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 630–639, 2014.     

Amino acid contribution to protein solubility: Asp, Glu, and Ser contribute more favorably than the other hydrophilic amino acids in RNase Sa

Poor protein solubility is a common problem in high-resolution structural studies, formulation of protein pharmaceuticals, and biochemical characterization of proteins. One popular strategy to improve protein solubility is to use site-directed mutagenesis to make hydrophobic to hydrophilic mutations on the protein surface. However, a systematic investigation of the relative contributions of all 20 amino acids to protein solubility has not been done. Here, 20 variants at the completely solvent-exposed position 76 of ribonuclease (RNase) Sa are made to compare the contributions of each amino acid. Stability measurements were also made for these variants, which occur at the i+1 position of a type II beta-turn. Solubility measurements in ammonium sulfate solutions were made at high positive net charge, low net charge, and high negative net charge. Surprisingly, there was a wide range of contributions to protein solubility even among the hydrophilic amino acids. The results suggest that aspartic acid, glutamic acid, and serine contribute significantly more favorably than the other hydrophilic amino acids especially at high net charge. Therefore, to increase protein solubility, asparagine, glutamine, or threonine should be replaced with aspartic acid, glutamic acid or serine.     

A Ca2+‐binding protein with numerous roles and uses: parvalbumin in molecular biology and physiology

Parvalbumins (PVs) are acidic, intracellular Ca²⁺‐binding proteins of low molecular weight. They are associated with several Ca²⁺‐mediated cellular activities and physiological processes. It has been suggested that PV might function as a “Ca²⁺ shuttle” transporting Ca²⁺ from troponin‐C (TnC) to the sarcoplasmic reticulum (SR) Ca²⁺ pump during muscle relaxation. Thus, PV may contribute to the performance of rapid, phasic movements by accelerating the contraction–relaxation cycle of fast‐twitch muscle fibers. Interestingly, PVs promote the generation of power stroke in fish by speeding up the rate of relaxation and thus provide impetus to attain maximal sustainable speeds. However, immunological monitoring of diverse tissues demonstrated that PVs are also present in non‐muscle cells. The axoplasmic transport and various intracellular secretory mechanisms including the endocrine secretions seem to be controlled by the Ca²⁺ regulation machinery. Any defect in the Ca²⁺ handling apparatus may cause several clinical problems; for instance, PV deficiency alters the neuronal activity, a key mechanism leading to epileptic seizures. Moreover, atypical relaxation of the heart results in diastolic dysfunction, which is a major cause of heart failure predominantly among the aged people. PV may offer a unique potential to correct defective relaxation in energetically compromised failing hearts through PV gene transfer. Consequently, PV gene transfer may present a new therapeutic approach to correct cellular disturbances in Ca²⁺ signaling pathways of diseased organs. Hence, PVs appear to be amazingly useful candidate proteins regulating a variety of cellular functions through action on Ca²⁺ flux management.     

Novel renin inhibitors containing derivatives of N‐alkylleucyl‐β‐hydroxy‐γ‐amino acids

In search for new drugs lowering arterial blood pressure, which could be applied in anti‐hypertensive therapy, research concerning agents blocking of renin‐angiotensin‐aldosteron system has been conducted. Despite many years of research conducted at many research centers around the world, aliskiren is the only one renin inhibitor, which is used up to now. Four novel potential renin inhibitors, having structure based on the peptide fragment 8–13 of human angiotensinogen, a natural substrate for renin, were designed and synthesized. All these inhibitors contain unnatural moieties that are derivatives of N‐methylleucyl‐β‐hydroxy‐γ‐amino acids at the P₂‐P₁' position: 4‐[N‐(N‐methylleucyl)‐amino]‐3‐hydroxy‐7‐(3‐nitroguanidino)‐heptanoic acid (AHGHA), 4‐[N‐(N‐methylleucyl)‐amino]‐3‐hydroxy‐5‐phenyl‐pentanoic acid (AHPPA) or 4‐[N‐(N‐methylleucyl)‐amino]‐8‐benzyloxycarbonylamino‐3‐hydroxyoctanoic acid (AAHOA). The previously listed synthetic β‐hydroxy‐γ‐amino acids constitute pseudodipeptidic units that correspond to the P₁‐P₁' position of the inhibitor molecule. An unnatural amino acid, 4‐methoxyphenylalanin (Phe(4‐OMe)), was introduced at the P₃ position of the obtained compounds. Three of these compounds contain isoamylamide of 6‐aminohexanoic acid (ε‐Ahx‐Iaa) at the P₂'‐P₃' position. The proposed modifications of the selected human angiotensinogen fragment are intended to increase bioactivity, bioavailability, and stability of the inhibitor molecule in body fluids and tissues. The inhibitor Boc‐Phe(4‐OMe)‐MeLeu‐AHGHA‐OEt was obtained in the form of an ethyl ester. The hydrophobicity coefficient, expressed as log P varied between 3.95 and 8.17. In vitro renin inhibitory activity of all obtained compounds was contained within the range 10^?6‐10^?9 M. The compound Boc‐Phe(4‐OMe)‐MeLeu‐AHPPA‐Ahx‐Iaa proved to be the most active (IC₅₀ = 1.05 × 10^?9 M). The compounds Boc‐Phe(4‐OMe)‐MeLeu‐AHGHA‐Ahx‐Iaa and Boc‐Phe(4‐OMe)‐MeLeu‐AHPPA‐Ahx‐Iaa are resistant to chymotrypsin. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Copper‐Chiral Camphor β‐Amino Alcohol Complex Catalyzed Asymmetric Henry Reaction

Four novel chiral amino alcohols were synthesized from D‐(+)‐camphor and utilized as ligands in a Cu(I)‐catalyzed asymmetric Henry reaction. The reactions were carried out under mild conditions with excellent enantioselectivities and moderate yields without the exclusion of air or moisture. The highest enantioselectivity was observed up to 94% enantiomeric excess (ee) with ligand L1 in toluene at room temperature. Chirality 27:761–765, 2015. © 2015 Wiley Periodicals, Inc.     

Synthesis,binding affinities and metabolic stability of dimeric dermorphin analogs modified with β3‐homo‐amino acids

In this study, proteinogenic amino acids residues of dimeric dermorphin pentapeptides were replaced by the corresponding β³‐homo‐amino acids. The potency and selectivity of hybrid α/β dimeric dermorphin pentapeptides were evaluated by competetive receptor binding assay in the rat brain using [3H]DAMGO (a μ ligand) and [3H]DELT (a δ ligand). Tha analog containing β³‐homo‐Tyr in place of Tyr (Tyr‐d ‐Ala‐Phe‐Gly‐β³‐homo‐Tyr‐NH‐)₂ showed good μ receptor affinity and selectivity (IC₅₀ = 0.302, IC₅₀ ratio μ/δ = 68) and enzymatic stability in human plasma. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

β‐Hairpin folding and stability: molecular dynamics simulations of designed peptides in aqueous solution

The structural properties of a 10‐residue and a 15‐residue peptide in aqueous solution were investigated by molecular dynamics simulation. The two designed peptides, SYINSDGTWT and SESYINSDGTWTVTE, had been studied previously by NMR at 278 K and the resulting model structures were classified as 3:5 β‐hairpins with a type I + G1 β‐bulge turn. In simulations at 278 K, starting from the NMR model structure, the 3:5 β‐hairpin conformers proved to be stable over the time period evaluated (30 ns). Starting from an extended conformation, simulations of the decapeptide at 278 K, 323 K and 353 K were also performed to study folding. Over the relatively short time scales explored (30 ns at 278 K and 323 K, 56 ns at 353 K), folding to the 3:5 β‐hairpin could only be observed at 353 K. At this temperature, the collapse to β‐hairpin‐like conformations is very fast. The conformational space accessible to the peptide is entirely dominated by loop structures with different degrees of β‐hairpin character. The transitions between different types of ordered loops and β‐hairpins occur through two unstructured loop conformations stabilized by a single side‐chain interaction between Tyr2 and Trp9, which facilitates the changes of the hydrogen‐bond register. In agreement with previous experimental results, β‐hairpin formation is initially driven by the bending propensity of the turn segment. Nevertheless, the fine organization of the turn region appears to be a late event in the folding process. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.     

Pyrazole amino acids: hydrogen bonding directed conformations of 3‐amino‐1H‐pyrazole‐5‐carboxylic acid residue

A series of model compounds containing 3‐amino‐1H‐pyrazole‐5‐carboxylic acid residue with N‐terminal amide/urethane and C‐terminal amide/hydrazide/ester groups were investigated by using NMR, Fourier transform infrared, and single‐crystal X‐ray diffraction methods, additionally supported by theoretical calculations. The studies demonstrate that the most preferred is the extended conformation with torsion angles ? and ψ close to ±180°. The studied 1H‐pyrazole with N‐terminal amide/urethane and C‐terminal amide/hydrazide groups solely adopts this energetically favored conformation confirming rigidity of that structural motif. However, when the C‐terminal ester group is present, the second conformation with torsion angles ? and ψ close to ±180° and 0°, respectively, is accessible. The conformational equilibrium is observed in NMR and Fourier transform infrared studies in solution in polar environment as well as in the crystal structures of other related compounds. The observed conformational preferences are clearly related to the presence of intramolecular interactions formed within the studied residue. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Non‐additive stabilization by halogenated amino acids reveals protein plasticity on a sub‐angstrom scale

It has been a long‐standing goal to understand the structure‐stability relationship of proteins, as optimal stability is essential for protein function and highly desirable for protein therapeutics. Halogenation has emerged as a minimally invasive strategy to probe the physical characteristics of proteins in solution, as well as enhance the structural stabilities of proteins for therapeutic applications. Although advances in synthetic chemistry and genetic code expansion have allowed for the rapid synthesis of proteins with diverse chemical sequences, much remains to be learned regarding the impact of these mutations on their structural integrity. In this contribution, we present a systematic study of three well‐folded model protein systems, in which their structural stabilities are assessed in response to various hydrogen‐to‐halogen atom mutations. Halogenation allows for the perturbation of proteins on a sub‐angstrom scale, offering unprecedented precision of protein engineering. The thermodynamic results from these model systems reveal that in certain cases, proteins can display modest steric tolerance to halogenation, yielding non‐additive consequences to protein stability. The observed sub‐angstrom sensitivity of protein stability highlights the delicate arrangement of a folded protein core structure. The stability data of various halogenated proteins presented herein should also provide guidelines for using halogenation as a strategy to improve the stability of protein therapeutics.