Side-chain Motions Research Articles

Wetting of a Dynamically Patterned Surface Is a Time-Dependent Matter.

In nature and many technological applications, aqueous solutions are in contact with patterned surfaces, which are dynamic over time scales spanning from ps to μs. For instance, in biology, exposed polar and apolar residues of biomolecules form a pattern, which fluctuates in time due to side chain and conformational motions. At metal/and oxide/water interfaces, the pattern is formed by surface topmost atoms, and fluctuations are due to, e.g., local surface polarization and rearrangements in the adsorbed water layer. All these dynamics have the potential to influence key processes such as wetting, energy relaxation, and biological function. Yet, their impact on the water H-bond network remains often elusive. Here, we leverage molecular dynamics to address this fundamental question at a self-assembled monolayer (SAM)/water interface, where ns dynamics is induced by frustrating SAM-water interactions via methylation of the terminal -OH groups of poly(ethylene glycol) (PEG) chains. We find that surface dynamics couples to the water H-bond network, inducing a response on the same ns time scale. This leads to time fluctuations of local wetting, oscillating from hydrophobic to hydrophilic environments. Our results suggest that rather than average properties, it is the local─ both in time and space─ solvation that determines the chemical-physical properties of dynamically patterned surfaces in water.

Analysis of chi angle distributions in free amino acids via multiplet fitting of proton scalar couplings.

Scalar couplings are a fundamental aspect of nuclear magnetic resonance (NMR) experiments and provide rich information about electron-mediated interactions between nuclei. 3 couplings are particularly useful for determining molecular structure through the Karplus relationship, a mathematical formula used for calculating 3 coupling constants from dihedral angles. In small molecules, scalar couplings are often determined through analysis of one-dimensional proton spectra. Larger proteins have typically required specialized multidimensional pulse programs designed to overcome spectral crowding and multiplet complexity. Here, we present a generalized framework for fitting scalar couplings with arbitrarily complex multiplet patterns using a weak-coupling model. The method is implemented in FitNMR and applicable to one-dimensional, two-dimensional, and three-dimensional NMR spectra. To gain insight into the proton-proton coupling patterns present in protein side chains, we analyze a set of free amino acid one-dimensional spectra. We show that the weak-coupling assumption is largely sufficient for fitting the majority of resonances, although there are notable exceptions. To enable structural interpretation of all couplings, we extend generalized and self-consistent Karplus equation parameterizations to all angles. An enhanced model of side-chain motion incorporating rotamer statistics from the Protein Data Bank (PDB) is developed. Even without stereospecific assignments of the beta hydrogens, we find that two couplings are sufficient to exclude a single-rotamer model for all amino acids except proline. While most free amino acids show rotameric populations consistent with crystal structure statistics, beta-branched valine and isoleucine deviate substantially.

Nanophase Segregation Drives Heterogeneous Dynamics in Amphiphilic PLMA‐b‐POEGMA Block‐Copolymers with Densely Grafted Architecture

AbstractThe self‐assembly and dynamics of amphiphilic diblock copolymers composed of densely grafted poly(oligo ethylene glycol methacrylate) (POEGMA) and poly(lauryl methacrylate) (PLMA) by means of calorimetry, small‐angle X‐ray scattering (SAXS), and dielectric spectroscopy are investigated. It is reported that the inherent immiscibility between the parent homopolymers, combined with the increased molar mass, results in strong segregation, maintained up to elevated temperatures (i.e., T = 423 K). SAXS reveals that well‐separated POEGMA and PLMA domains self‐assemble into spheres with bicontinuous cubic packing and in lamellar nanostructure in copolymers with respective compositions of 16 and 52 wt.% PLMA. This strong segregation enables the weak crystallization/melting of the short ethylene glycol chains. Additionally, molecular dynamics are investigated through isothermal dielectric and calorimetry measurements. The segmental dynamics (i.e., Tg‐related) of POEGMA and PLMA closely resemble that found in respective homopolymers, implying heterogeneous dynamics. In the glassy state, the local motions of the POEGMA side chains predominantly govern the observed secondary processes in the copolymers. The results on the heterogeneous dynamics in the current amphiphilic diblock copolymers with the densely grafted architecture are compared and contrasted with copolymers having a bottle–brush architecture lacking the amphiphilic nature.

Quasielastic neutron scattering study on low-hydrated myoglobin inside silica nanopores

In this study, nanosecond-scale dynamics of low-hydrated myoglobin (Mb) encapsulated in mesoporous silica (MPS) with 7.8 nm pores was investigated by using high resolution (energy resolution: 1 μeV) neutron backscattering spectroscopy. The apparent hydration level of the encapsulated myoglobin was 0.06, which was deduced by assuming a uniform distribution of water within myoglobin encapsulated mesoporous silica. The mean square displacement of myoglobin derived from an elastic fixed-window scan was essentially proportional to temperature in the range 120–300 K, indicating the absence of dynamical transition induced by the onset of translational diffusion in the hydration D2O. Since the width of quasielastic neutron scattering (QENS) peaks of the encapsulated Mb was Q-independent at 220, 240, and 265 K, the observed nanosecond-scale dynamics was attributed to internal motions of the encapsulated Mb, such as slow motions of amino acid groups or side chains. The fraction of immobile H-atoms in Mb was estimated to be above 0.8, which was larger than that for a free protein with a similar molecular weight to Mb. The large immobile fraction indicates that the nanosecond-scale motions of amino acid residues and side chains in the vicinity of the pore wall are restricted by the interfacial interactions with the rigid pore wall.

Analytical Framework to Understand the Origins of Methyl Side-Chain Dynamics in Protein Assemblies.

Side-chain motions play an important role in understanding protein structure, dynamics, protein-protein, and protein-ligand interactions. However, our understanding of protein side-chain dynamics is currently limited by the lack of analytical tools. Here, we present a novel analytical framework employing experimental nuclear magnetic resonance (NMR) relaxation measurements at atomic resolution combined with molecular dynamics (MD) simulation to characterize with a high level of detail the methyl side-chain dynamics in insoluble protein assemblies, using amyloid fibrils formed by the prion HET-s. We use MD simulation to interpret experimental results, where rotameric hops, including methyl group rotation and χ1/χ2 rotations, cannot be completely described with a single correlation time but rather sample a broad distribution of correlation times, resulting from continuously changing local structure in the fibril. Backbone motion similarly samples a broad range of correlation times, from ∼100 ps to μs, although resulting from mostly different dynamic processes; nonetheless, we find that the backbone is not fully decoupled from the side-chain motion, where changes in side-chain dynamics influence backbone motion and vice versa. While the complexity of side-chain motion in protein assemblies makes it very challenging to obtain perfect agreement between experiment and simulation, our analytical framework improves the interpretation of experimental dynamics measurements for complex protein assemblies.

Unraveling motion in proteins by combining NMR relaxometry and molecular dynamics simulations: A case study on ubiquitin.

Nuclear magnetic resonance (NMR) relaxation experiments shine light onto the dynamics of molecular systems in the picosecond to millisecond timescales. As these methods cannot provide an atomically resolved view of the motion of atoms, functional groups, or domains giving rise to such signals, relaxation techniques have been combined with molecular dynamics (MD) simulations to obtain mechanistic descriptions and gain insights into the functional role of side chain or domain motion. In this work, we present a comparison of five computational methods that permit the joint analysis of MD simulations and NMR relaxation experiments. We discuss their relative strengths and areas of applicability and demonstrate how they may be utilized to interpret the dynamics in MD simulations with the small protein ubiquitin as a test system. We focus on the aliphatic side chains given the rigidity of the backbone of this protein. We find encouraging agreement between experiment, Markov state models built in the χ1/χ2 rotamer space of isoleucine residues, explicit rotamer jump models, and a decomposition of the motion using ROMANCE. These methods allow us to ascribe the dynamics to specific rotamer jumps. Simulations with eight different combinations of force field and water model highlight how the different metrics may be employed to pinpoint force field deficiencies. Furthermore, the presented comparison offers a perspective on the utility of NMR relaxation to serve as validation data for the prediction of kinetics by state-of-the-art biomolecular force fields.

Dynamics of activation in the voltage-sensing domain of Ciona intestinalis phosphatase Ci-VSP

The Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) is a membrane protein containing a voltage-sensing domain (VSD) that is homologous to VSDs from voltage-gated ion channels responsible for cellular excitability. Previously published crystal structures of Ci-VSD in putative resting and active conformations suggested a helical-screw voltage sensing mechanism in which the S4 helix translocates and rotates to enable exchange of salt-bridge partners, but the microscopic details of the transition between the resting and active conformations remained unknown. Here, by combining extensive molecular dynamics simulations with a recently developed computational framework based on dynamical operators, we elucidate the microscopic mechanism of the resting-active transition at physiological membrane potential. Sparse regression reveals a small set of coordinates that distinguish intermediates that are hidden from electrophysiological measurements. The intermediates arise from a noncanonical helical-screw mechanism in which translocation, rotation, and side-chain movement of the S4 helix are only loosely coupled. These results provide insights into existing experimental and computational findings on voltage sensing and suggest ways of further probing its mechanism.

Multistep conformational changes leading to the gate opening of light-driven sodium pump rhodopsin

Membrane transport proteins require a gating mechanism that opens and closes the substrate transport pathway to carry out unidirectional transport. The "gating" involves large conformational changes and is achieved via multistep reactions. However, these elementary steps have not been clarified for most transporters due to the difficulty of detecting the individual steps. Here, we propose these steps for the gate opening of the bacterial Na+ pump rhodopsin, which outwardly pumps Na+ upon illumination. We herein solved an asymmetric dimer structure of Na+ pump rhodopsin from the bacterium Indibacter alkaliphilus. In one protomer, the Arg108 sidechain is oriented toward the protein center and appears to block a Na+ release pathway to the extracellular (EC) medium. In the other protomer, however, this sidechain swings to the EC side and then opens the release pathway. Assuming that the latter protomer mimics the Na+-releasing intermediate, we examined the mechanism for the swing motion of the Arg108 sidechain. On the EC surface of the first protomer, there is a characteristic cluster consisting of Glu10, Glu159, and Arg242 residues connecting three helices. In contrast, this cluster is disrupted in the second protomer. Our experimental results suggested that this disruption is a key process. The cluster disruption induces the outward movement of the Glu159-Arg242 pair and simultaneously rotates the seventh transmembrane helix. This rotation resultantly opens a space for the swing motion of the Arg108 sidechain. Thus, cluster disruption might occur during the photoreaction and then trigger sequential conformation changes leading to the gate-open state.

Polymer-in-Salt Electrolyte from Poly(caprolactone)-graft-polyrotaxane for Ni-Rich Lithium Metal Batteries at Room Temperature.

Solid-state lithium batteries with solid polymer electrolytes have recently attracted extensive attention due to their promising potential in high energy density and safety. However, the principal issues plaguing the solid polymer electrolytes are their restricted ionic conductivities at ambient temperature and the limited tolerance to the widely used high-voltage cathodes (such as LiNi0.8Mn0.1Co0.1O2, NCM811), thus limiting their practical applications seriously. In this regard, a superior polymer-in-salt solid electrolyte from poly(caprolactone)-graft-polyrotaxane (PGPE) is developed for high-voltage lithium batteries operated at room temperature. The PGPE displays remarkable electrochemical properties at room temperature, with an exceptional ionic conductivity of 4.89 × 10-4 S cm-1 and a lithium-ion transference number of approximately 0.64, stemming from the rapid segmental motions of PCL sidechains by the enhanced dynamics of the cyclic molecules along the axial polymer chain of polyrotaxane. More importantly, the PGPE demonstrates a high electrochemical oxidation voltage of ∼4.7 V, suggesting the excellent electrochemical stability of PGPE against the NCM811-based cathode. Owing to the dense LiF-rich CEI self-generated on the NCM811 particles in the cathode, the transition metal ion diffusion is successfully constrained and the PGPE is well protected from continuous decomposition. The PGPE also shows superior interfacial stability between the metallic Li and the electrolyte. As a result, the all-solid-state NCM811|PGPE|Li cell exhibits superior discharge capacity (196 mAh g-1) and extraordinary long-term cycling stability (74% capacity retention at 150 cycles) at 30 °C.

Influence of mixed-frequency medium-voltage and environmental stress on the aging of epoxy

Recent developments in power electronic technologies lead to new challenges for insulation systems. This contribution aims to clarify the influence of a broad range of mixed-frequency (MF) medium-voltage and environmental stress parameters on the aging of epoxy insulation. For this purpose, test samples are stressed with an AC (50 Hz) or a DC voltage, superimposed with a pulse-width-modulated (PWM) voltage (kHz range). An analysis of the samples’ health state is carried out after the aging by the evaluation of potential aging markers (AC breakdown strength, dielectric permittivity, glass transition temperature, Fourier-transform infrared spectroscopy spectra). Although the main focus of this work is on aging below the inception of partial discharges (PDs), it was first confirmed that PD-related aging depends mainly on the peak voltage stress. In contrast, the results obtained by aging below PD inception suggests a dependence on the root-mean-square of the applied voltage stress, and consequently on the energy dissipation. Aging in the PD-free regime was only observed at alternating electric field stress and high relative humidity or elevated temperatures. No influences of space charge and of the slew rate of the PWM voltage were observed. Remarkably, higher PWM frequencies lead to less insulation aging. This might be attributed to the increasing hindrance of polymer side chain movement at higher frequencies, as observed by dielectric spectroscopy. In addition, it is indicated that the aging mechanisms under MF voltage stress result from superimposed single-frequency aging mechanisms and that aging is activated after a latency period. Of the investigated potential aging markers, only the residual breakdown strength revealed aging effects, which correlates with lifetime observations in the PD-free voltage stress regime. It is hypothesized that the aging mechanism is associated with a rearrangement of the free volume in the polymer, followed by a localized breaking of van der Waals bonds.

TinyIFD: A High-Throughput Binding Pose Refinement Workflow Through Induced-Fit Ligand Docking.

A critical step in structure-based drug discovery is predicting whether and how a candidate molecule binds to a model of a therapeutic target. However, substantial protein side chain movements prevent current screening methods, such as docking, from accurately predicting the ligand conformations and require expensive refinements to produce viable candidates. We present the development of a high-throughput and flexible ligand pose refinement workflow, called "tinyIFD". The main features of the workflow include the use of specialized high-throughput, small-system MD simulation code mdgx.cuda and an actively learning model zoo approach. We show the application of this workflow on a large test set of diverse protein targets, achieving 66% and 76% success rates for finding a crystal-like pose within the top-2 and top-5 poses, respectively. We also applied this workflow to the SARS-CoV-2 main protease (Mpro) inhibitors, where we demonstrate the benefit of the active learning aspect in this workflow.

Boronic Ester Transesterification Accelerates Ion Conduction for Comb-like Solid Polymer Electrolytes

Polyether is a kind of ideal polymer matrix for solid polymer electrolytes (SPEs), but its inherent drawbacks (such as the semi-crystalline nature and strong complexation of lithium ions) still hinder their commercial application. The construction of comb-like SPEs effectively increases their functionalities and improves the comprehensive performance. Herein, we apply a dynamic boronic ester exchange strategy to simultaneously enhance the lithium-ion conduction and self-healing ability of the SPEs, thus effectively solving the current problems of polyether. The copolymer (GPA) containing side chains with poly(ethylene glycol) (PEG) and diol groups was obtained through reversible addition–fragmentation chain-transfer (RAFT) polymerization and hydrolysis, and PBA-M2070 containing the boronic acid group and flexible polyether segments was obtained via Schiff base reaction. Boronic ester bonds were formed by dehydration of the diol groups of GPA and the boronic acid groups of PBA-M2070. Dynamic exchange of the boronic ester bonds at the branched sites enhanced the side-chain motion, thus increasing the self-healing ability and electrochemical performance of the SPEs, as well as the cycling stability of assembled batteries. Benefiting from the dynamic boronic ester exchange and enhanced side-chain mobility, the SPEs exhibited self-healing within 10 min and an ionic conductivity of 1.58 × 10–5 S cm–1 at 30 °C. The assembled lithium metal batteries (LMBs) were operated stably at 0.5C for 300 cycles. The dynamic boronic ester exchange strategy used to enhance the LMBs properties provides new insight into the design of comb-like polymer electrolytes.

Precisely Controllable Artificial Muscle with Continuous Morphing based on "Breathing" of Supramolecular Columns.

Skeletal muscles are natural motors executing sophisticated work through precise control of linear contraction. Although various liquid crystal polymersbased artificial muscles havebeen designed, the mechanism based on mainly the order-disorder transition usually leads to discrete shape morphing, leaving arbitrary and precise deformation a huge challenge. Here, one novel photoresponsive hemiphasmidic side-chain liquid crystal polymer with a unique "breathing" columnar phase that enables continuous morphing is presented. Due to confinement inside the supramolecular columnar assembly, the cooperative movements of side-chains and backbones generate a significant negative thermal expansion and lead to temperature-controllable muscle-like elongation/contraction in the oriented polymer strip. The irreversible isomerization of the photoresponsive mesogens results in the synergistic phototunable bending and high-contrast fluorescence change. Based on the orthogonal responses to heat and light, controllable arm-like bending motions of this material, which is applicable in constructing advanced artificial muscles or intelligent soft robotics, are further demonstrated.

Mechanical and thermal properties of carbon fiber epoxy composite with interlaminar graphene at elevated temperature

In this study we examined the mechanisms of graphene reinforcement in CFRP and the resulting improvements to mechanical strength, resistance to crack propagation, and thermal conductivity at elevated temperatures. Plant-based graphene nanoplatelets (pGNP), produced from renewable biomass and with flakes 3–10 layers dispersed in alcohol/water was applied via spray at 2.3 g/m2 to unidirectional carbon fiber/epoxy prepreg, creating an interlaminar nanocomposite. Raman spectroscopy, XPS, and DMA show polymer crosslinking with graphene surface groups and the resulting restriction of side chain movement. These restrictions improve composite performance at ambient and elevated temperatures, extending the damage process zone and increasing fracture toughness. Interlaminar pGNP improved Mode I fracture toughness at crack initiation by 145% at 20 °C and 126% at 90 °C, with fracture toughness improved by 53% and 52% during propagation at 20 °C and 90 °C, respectively. Mode II fracture toughness was not changed at 20 °C and improved 55% at 90 °C.

Reaction Dynamics in the Chrimson Channelrhodopsin: Observation of Product-State Evolution and Slow Diffusive Protein Motions.

Chrimson is a red-light absorbing channelrhodopsin useful for deep-tissue optogenetics applications. Here, we present the Chrimson reaction dynamics from femtoseconds to seconds, analyzed with target analysis methods to disentangle spectrally and temporally overlapping excited- and product-state dynamics. We found multiple phases ranging from ≈100 fs to ≈20 ps in the excited-state decay, where spectral features overlapping with stimulated emission components were assigned to early dynamics of K-like species on a 10 ps time scale. Selective excitation at the maximum or the blue edge of the absorption spectrum resulted in spectrally distinct but kinetically similar excited-state and product-state species, which gradually became indistinguishable on the μs to 100 μs time scales. Hence, by removing specific protein conformations within an inhomogeneously broadened ensemble, we resolved slow protein backbone and amino acid side-chain motions in the dark that underlie inhomogeneous broadening, demonstrating that the latter represents a dynamic interconversion between protein substates.

Conformational Dynamics of mCherry Variants: A Link between Side-Chain Motions and Fluorescence Brightness.

The 3-fold higher brightness of the recently developed mCherry-XL red fluorescent protein (FP) compared to its progenitor, mCherry, is due to a significant decrease in the nonradiative decay rate underlying its increased fluorescence quantum yield. To examine the structural and dynamic role of the four mutations that distinguish the two FPs and closely related variants, we employed microsecond time scale, all-atom molecular dynamics simulations. The simulations revealed that the I197R mutation leads to the formation of multiple hydrogen-bonded contacts and increased rigidity of the β-barrel. In particular, mCherryXL showed reduced nanosecond time scale breathing of the gap between the β7 and β10-strands, which was previously shown to be the most flexible region of mCherry. Together with experimental results, the simulations also reveal steric interactions of residue 161 and a network of hydrogen-bonding interactions of the chromophore with residues at positions 59, 143, and 163 that are critical in perturbing the chromophore electronic structure. Finally, we shed light on the conformational dynamics of the conserved residues R95 and S146, which are hydrogen-bonded to the chromophore, and provide physical insights into the observed photophysics. To the best of our knowledge, this is the first study that evaluates the conformational space for a set of closely related FPs generated by directed evolution.

Polymeric Single-Ion Conductors with Enhanced Side-Chain Motion for High-Performance Solid Zinc-Ion Batteries.

Zn-based solid polymer electrolytes (SPEs) have enormous potential in realizing high-performance zinc-ion batteries. Polymeric single-ion conductor (PSIC)-based SPEs can largely eradicate anion migration and side reactions of electrodes with decreased polarization, but the ionic conductivity is still unsatisfactory due to the tight localized ion interactions and sluggish chain motion. Herein, by employing the heterocyclic tetrazole as the anionic center of the side chain, a novel PSIC is fabricated with optimized charge delocalization and enhanced side-chain motion. The as-prepared PSIC delivers an ionic conductivity up to 5.4 × 10-4 S cm-1 with an ultrahigh Zn2+ transference number of 0.94. Based on the PSIC, dendrite-free and hydrogen-free Zn plating/stripping cycling (2000h) is achieved. A further assembled Zn‖V2 O5 battery exhibits superior performances to other solid ZIBs, including a high discharge capacity, excellent rate capability, and long cycling life. In addition, a remarkable shelf-life (90 d), low self-discharge rate, and good temperature adaptability of the solid battery can be achieved benefiting from the high stability of the SPE during operation. The PSIC-based SPEs with advanced ion-transport structure endow solid ZIBs with significant performance improvement, high safety, and durability.

Explicit models of motions to analyze NMR relaxation data in proteins.

Nuclear Magnetic Resonance (NMR) is a tool of choice to characterize molecular motions. In biological macromolecules, pico- to nanosecond motions, in particular, can be probed by nuclear spin relaxation rates, which depend on the time fluctuations of the orientations of spin interaction frames. For the past 40 years, relaxation rates have been successfully analyzed using the Model-Free (MF) approach, which makes no assumption on the nature of motions and reports on the effective amplitude and timescale of the motions. However, obtaining a mechanistic picture of motions from this type of analysis is difficult at best, unless complemented with molecular dynamics (MD) simulations. In spite of their limited accuracy, such simulations can be used to obtain the information necessary to build explicit models of motions designed to analyze NMR relaxation data. Here, we present how to build such models, suited in particular to describe motions of methyl-bearing protein side chains and compare them with the MF approach. We show on synthetic data that explicit models of motions are more robust in the presence of rotamer jumps which dominate the relaxation in methyl groups of protein side chains. We expect this work to motivate the use of explicit models of motion to analyze MD and NMR data.

Structure of lactate oxidase from Enterococcus hirae revealed new aspects of active site loop function: Product-inhibition mechanism and oxygen gatekeeper.

l-Lactate oxidase (LOx) is a flavin mononucleotide (FMN)-dependent triose phosphate isomerase (TIM) barrel fold enzyme that catalyzes the oxidation of l-lactate using oxygen as a primary electron acceptor. Although reductive half-reaction mechanism of LOx has been studied by structure-based kinetic studies, oxidative half-reaction and substrate/product-inhibition mechanisms were yet to be elucidated. In this study, the structure and enzymatic properties of wild-type and mutant LOxs from Enterococcus hirae (EhLOx) were investigated. EhLOx structure showed the common TIM-barrel fold with flexible loop region. Noteworthy observations were that the EhLOx crystal structures prepared by co-crystallization with product, pyruvate, revealed the complex structures with "d-lactate form ligand," which was covalently bonded with a Tyr211 side chain. This observation provided direct evidence to suggest the product-inhibition mode of EhLOx. Moreover, this structure also revealed a flip motion of Met207 side chain, which is located on the flexible loop region as well as Tyr211. Through a saturation mutagenesis study of Met207, one of the mutants Met207Leu showed the drastically decreased oxidase activity but maintained dye-mediated dehydrogenase activity. The structure analysis of EhLOx Met207Leu revealed the absence of flipping in the vicinity of FMN, unlike the wild-type Met207 side chain. Together with the simulation of the oxygen-accessible channel prediction, Met207 may play as an oxygen gatekeeper residue, which contributes oxygen uptake from external enzyme to FMN. Three clades of LOxs are proposed based on the difference of the Met207 position and they have different oxygen migration pathway from external enzyme to active center FMN.

Deep learning approaches for conformational flexibility and switching properties in protein design.

Following the hugely successful application of deep learning methods to protein structure prediction, an increasing number of design methods seek to leverage generative models to design proteins with improved functionality over native proteins or novel structure and function. The inherent flexibility of proteins, from side-chain motion to larger conformational reshuffling, poses a challenge to design methods, where the ideal approach must consider both the spatial and temporal evolution of proteins in the context of their functional capacity. In this review, we highlight existing methods for protein design before discussing how methods at the forefront of deep learning-based design accommodate flexibility and where the field could evolve in the future.